elucidation for gastrokinetic effects of a novel 5-ht...

Elucidation for Gastrokinetic Effects of a Novel 5-HT4 Receptor

Agonist, CJ-033466 and its Related Pharmacological Characterization

of 5-HT Receptor Subtypes

2007

Tohru Komada

Contents Abbreviations ……………………………………………………………….……. 1 General Remarks ……………………………………………………..………..…. 2 Chapter I: Effects of 5-HT4 receptor agonists on gastric motility in dogs

1. Introduction …………………………………………………………. 6 2. Materials and Methods ……………………………………………… 7 3. Results ………………………………………………………………. 9 4. Discussion …………………………………………………………… 21

Chapter II: Effects of 5-HT4 receptor agonists on gastric emptying and gastric motility in rats

1. Introduction …………………………………………………………. 23 2. Materials and Methods ……………………………………………… 23 3. Results ………………………………………………………………. 25 4. Discussion …………………………………………………………... 43

Chapter III: Pharmacological characterization of 5-HT-receptor subtypes in circular muscle from the rat stomach

1. Introduction …………………………………………………………. 45 2. Materials and Methods ……………………………………………… 45 3. Results ………………………………………………………………. 47 4. Discussion ………………………………………………………...… 58

Chapter IV: Effects of 5-HT4 receptor agonists on contractile responses of circular muscle from the rat stomach

1. Introduction …………………………………………………………. 63 2. Materials and Methods ……………………………………………… 63 3. Results ………………………………………………………………. 64 4. Discussion …………………………………………………………... 78

Chapter V: Effects of CJ-033466 on ACh release from human gastric antral tissues 1. Introduction …………………………………………………………. 80 2. Materials and Methods ……………………………………………… 80 3. Results ………………………………………………………………. 82 4. Discussion …………………………………………………………... 87

Concluding Remarks ……………………………………………………………... 88 Acknowledgements ………………………………………………….…………… 89 References …………..……………………………………………………………. 90 List of publications ………………………….…………………………………….. 100 Referees …………………………………………………………………………… 101

Abbreviations ACh : acetylcholine α-methyl-5-HT : α-methyl-5-hydroxytryptamine CCh : carbachol EFS : electrical field stimulation ES : electrical stimulation GERD : gastroesophageal reflux disease HERG : human ether-a-go-go-related gene 5-HT : 5-hydroxytryptamine IMC : interdigestive migrating contraction IBS : irritable bowel syndrome LES : lower esophageal sphincter L-NOARG : Nω-Nitro-L-arginine MC : methylcellulose NO : nitric oxide SBECD : sulfobutylether-β-cyclodextrin TTX : tetrodotoxin TLESR : transient lower esophageal sphincter relaxation TMM : tunica muscularis mucosae

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General Remarks Serotonin (5-hydroxytryptamine, 5-HT) is an endogenous monoamine which has a wide range of physiological actions. Originally, Erspamer et al. (1) discovered a smooth muscle contracting substance “enteramine”, named because large amounts of it were stored in enterochromaffin cells of the gastrointestinal tract. Independently, Rapport et al. (2) identified the serum vasoconstrictor factor releasing from platelets during clotting of blood as “serotonin”. In 1952, enteramine was found to be identical to the vasoconstrictor substance known as serotonin (3). The gastrointestinal tract is the main source of serotonin in the body (4). More than 90% of serotonin in the body exists in the enterochromaffin cells of the gut mucosa. It is now recognized that 5-HT is also contained in the enteric nervous systems of the gastrointestinal tract (5). Serotonin works as a neurotransmitter and a paracrine signaling molecule and exerts a variety of effects on intrinsic enteric neurons, extrinsic afferent neurons, enterocytes and smooth muscle cells. They express multiple 5-HT receptor types and subtypes regulating gastrointestinal motility (6), secretion (7) and perception (8). 5-HT receptor subtypes have been classified into seven main types as 5-HT1, 5-HT2, 5-HT3, 5-HT4, 5-ht5, 5-HT6 and 5-HT7. Furthermore, 5-HT1, 5-HT2 and 5-ht5 receptors have been subdivided into 5-HT1A, 5-HT1B, 5-HT1D, 5-HT1E, 5-HT1F, 5-HT2A, 5-HT2B, 5-HT2C, 5-ht5A and 5-ht5B (9-11). Recently, 5-HT1E, 5-HT1F, 5-ht5, 5-HT6 and 5-HT7 receptors subtypes have been characterized using molecular cloning techniques. Therefore, these receptors have not been extensively characterized pharmacologically, except for the 5-HT7 receptor. With the exception of the 5-HT3 receptor, which is a ligand-gated ion channel, 5-HT receptors belong to the G-protein-coupled receptor superfamily. It is generally accepted that the 5-HT1A, 5-HT1B, 5-HT1D, 5-HT2A, 5-HT2B, 5-HT3, 5-HT4 and 5-HT7 receptor subtypes are known to affect the gut function (12, 13). 5-HT4 receptors couple to Gs protein and promote cAMP formation.

5-HT is thought to be involved in the pathophysiology of several clinical entities, including functional gut disorders such as irritable bowel syndrome (IBS), functional dyspepsia, chronic constipation, carcinoid diarrhea, and chemotherapy induced emesis (14-17). The discovery of selective 5-HT receptor agonists and antagonists brought up intense interest of its application on gastrointestinal pharmacotherapy. 5-HT3 receptor antagonists demonstrate the potential therapeutic role for diarrhea-predominant IBS, carcinoid diarrhea, and chemotherapy induced emesis (18-20). 5-HT4 receptor agonists demonstrate the potential therapeutic role for constipation-predominant IBS (c-IBS) and

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chronic constipation (21). 5-HT1 receptor agonists, sumatriptan (5-HT1B/1D receptor agonist) and buspiron (5-HT1A receptor agonist) enhanced gastric accommodation, indicating the therapeutic role for functional dyspepsia (22, 23).

Furthermore, the pathophysiology of gastroesophageal reflux disease (GERD) is closely related to gastric dysmotiltiy. Transient lower esophageal sphincter relaxation (TLESR) is the most important mechanism of reflux in GERD. The induction of TLESRs in patients with GERD is primarily related to stimulation of mechanoreceptors in the proximal stomach. Therefore, delayed gastric emptying or altered fundic accommodation may contribute to increase triggering of TLESRs.

5-HT4 agonists such as cisapride, mosapride and tegaserod are known to have gastroprokinetic effects and restore gastrointestinal dysmotility in subjects. It is reported that 5-HT4 agonists act through enhanced release of acetylcholine via 5-HT4 receptors at the postganglionic level result in the acceleration of gastrointestinal motility (24-26).

Cisapride with a benzamide structure was released in the United States as Propulsid in 1993 and is available as Prepulsid in Europe and other countries. This agent is the first panprokinetic developed. Cisapride stimulates esophageal peristalsis, lower esophageal sphincter (LES) pressure, and gastric emptying (27-29), which explains its efficacy in GERD. This reducing gastric distention can lead to fewer TLESRs and less gastroesophageal reflux. Cisapride had also used for a variety of gastrointestinal complaints, including functional constipation, c-IBS and diabetic gastroparesis. However, cisapride was withdrawn from the market in 2000 due to its cardiovascular side effects (30).

Mosapride with a benzamide structure was released in Japan as Gasmotin in 1998 and also in South Korea and China. Mosapride has been used for the treatment of gastrointestinal symptoms as heartburn, nausea and vomiting with chronic gastritis. Early clinical study indicated that mosapride promoted gastric emptying in healthy volunteers (31), enhanced gastric motility in diabetes (32). Ruth et al. (33) reported inhibition of GERD symptoms in 1998, but no further work has been reported in this area.

Tegaserod with an aminoguanidine indole structure was released in Mexico as Zelnorm in 2001 and is available in other countries except for Western Europe for c-IBS and chronic constipation. Tegaserod also demonstrated the acceleration of gastric emptying in healthy volunteers (34) and the reduction of postprandial esophageal acid exposure result from decreased TLESR in GERD patients (35).

Although mosapride and tegaserod demonstrated gastroprokinetic effects, their indications are limited compared with those of cisapride. Therefore, there are unmet

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needs in clinical therapy after the withdrawal of cisapride. A novel 5-HT4 agonist, CJ-033466 has been developed to fill the gap between them. CJ-033466 is an imidazopyridine derivative which is a different structure class from cisapride, mosapride and tegaserod. The isolated rat tunica muscularis mucosae (TMM) has proved to be useful in characterizing the pharmacology of 5-HT4 receptor agonists (36, 37). In this functional assay, EC50 values of cisapride, mosapride, tegaserod and CJ-033466 for 5-HT4 receptors are 49, >1000, 0.15 and 1.3 nM, respectively (Fig. 1). Other than 5-HT4 agonism, it is reported that cisapride had 5-HT1 and 5-HT3 antagonism (25), a metabolite of mosapride had 5-HT3 antagonism (38), and tegaserod had 5-HT2B antagonism (39).

Therefore, the present study aims to profile the gastroprokinetic effect and the pharmacological characterization of CJ-033466 in in vivo canine gastric motility, rat gastric emptying and motility models and in vitro rat isolated circular muscle from the stomach compared with those of cisapride, mosapride and tegaserod.

Regional and functional differences of 5-HT receptor subtypes in the rat stomach remains unclear except for 5-HT2B receptors on the fundus. Therefore, pharmacological characterization of 5-HT receptor subtypes in circular muscle from the antrum, corpus and fundus of rats were studied for clarifying the similarity and difference among the 5-HT4 receptor agonists.

Finally, the effect of CJ-033466 on acetylcholine release from human gastric antral tissues was evaluated in order to extrapolate the activity of CJ-033466 in humans. All animal experiments used in the present study (Chapter I to IV) were approved by the Animal Ethics Committee at the Nagoya Laboratories of Pfizer Global Research and Development according to the Laboratory Animal Welfare guidelines. All human tissue experiments in the present study (Chapter V) were approved by The Ethical Committee of Nagasaki University School of Medicine, based on the informed consent of patients.

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Cl

H2N

NH

O

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BenzamideRat TMM: EC50 = 49 nM

Emax = 57 %

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BenzamideRat TMM: EC50 > 1000 nM

Emax: 50 % @ 10 µM

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Aminoguanidine indoleRat TMM: EC50 = 0.15 nM

Emax = 76 %

N NH2N

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NH N

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ImidazopyridineRat TMM: EC50 = 1.3 nM

Emax = 58 %

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CJ-033466


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N NH2N

ClO

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CJ-033466


Emax = 58 %

Figure 1. Chemical structures, potencies and efficacies of 5-HT4 agonists, cisapride, mosapride, tegaserod and CJ-033466. The potency and efficacy were examined with rat tunica muscularis mucosae (TMM).

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Chapter I: Effects of 5-HT4 receptor agonists on gastric motility in dogs 1. Introduction

The dog stomach is anatomically similar to that of human. The patterns of gastrointestinal contractions in the interdigestive (fasted) and the digestive (postprandial) states of dogs are homologous to human (40, 41). Canine gastric motility and gastric emptying models are widely used to study the physiology of gastrointestinal tract and the effect of gastrokinetics on the stomach. It is known that the data from dogs are well extrapolated to human.

The contractile patterns before and after feeding in dogs are quite different, which is also seen in the human gastrointestinal tract (42). In the fasted state, a group of intense phasic contractions occur in the stomach at about 90 min intervals and last for about 20 min and then stop abruptly and spontaneously. These grouped, strong phasic contractions appear to immigrate along the small intestine in a caudal direction at a constant velocity, which called interdigestive migrating contraction (IMC) (43). It is generally accepted that motilin released in the fasted state induces IMC in dogs and humans (44). The fasted state is by no means a quiescent state after digestion and absorption are terminated, even then, desquamated epithelial cells, mucus, and saliva may accumulate in the stomach. The strong contractions are considered to move them into and down the small bowel and into the large bowel. Therefore, the IMC are regarded as the interdigestive ‘housekeeper’ of the gut. On the other hand, in the postprandial state, the gastric contractions are regular and continuous. The contraction force during the postprandial state is about one-third to one-fourth of that of the interdigestive contractions. After the ingestion of food, the contractile activity of the gastric body decreases to accommodate the food in the stomach. It has been called ‘receptive relaxation’. In association with the receptive relaxation of the gastric body, regular and continuous contractions start to occur in the gastric antrum. These contractions grind and mix food with gastric secretion during the initial period and evacuate the mixture into the duodenum during the latter period. In the postprandial state, peptides such as gastrin, VIP, somatostatin and CCK are released from the stomach and duodenum and regulate gastric motility directly and indirectly (45-48).

The dog gastric motility was measured using a strain gauge force transducer method. Strain gauge transducers have been used to record sustained contractions and relaxations as well as circular and longitudinal muscle phasic activity. When sewn along with the circular muscle of the stomach, the transducer records phasic contractions

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which may be superimposed on tonal changes of circular muscle activity. The strain gauge transducer method has several advantages over other methods of gastrointestinal motility recording. The unit directly records the contractile activity of the smooth muscle. Strain gauge transducers are sewn on the serosal surface of the gastrointestinal tract. Therefore, there is no artificial effect on intraluminal conditions such as an interference with flow of intraluminal contents and stimulation of mucosal receptors. Since they are permanently fixed in position, the same site can be monitored repeatedly without using elaborated procedures to place the sensors in the desired position. Furthermore, it is well-known that gastrointestinal motility is influenced with anesthetics and under the stress. With telemetry system, gastrointestinal motility can be measured under fully conscious and minimal restraint conditions.

In this chapter, considering the difference with the contractile pattern and mechanisms involving in the fasted and postprandial state, effects of 5-HT4 agonists were examined using the strain gauge force transducer method under the fasted and postprandial conditions in dogs. 2. Materials and Methods Animals

Male beagle dogs weighing 9.9-14.7 kg (Oriental Yeast Co., Ltd., Tokyo, Japan) were used. The animals were acclimatized to the laboratory conditions during experiments. Preparation of animals

Dogs were anesthetized with isoflurane and the abdominal cavity was opened under aseptic conditions. Extraluminal force transducers (F-12IS, Star Medical, Tokyo, Japan) were sutured onto the seromuscular layer of the gastric antrum, (3 cm proximal to the pyloric ring), the gastric body (10-15 cm proximal to the pyloric ring), the duodenum (10 cm distal to the pyloric ring), and the proximal colon (5 cm distal to the ileocecum). The lead wires of these transducers were taken out of the abdominal cavity and then brought out through a skin incision made between the scapulae. After surgery, protective jackets were placed on the dogs, and they were housed in individual cages. Recording of the gastric motility was started at least 2 weeks after surgery. Experiments in the fasted state

After an overnight fast, the dogs were placed in the shield room, and recording of gastrointestinal motility in the fasted state initiated. Gut motility was measured with a

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telemetry system (GTS-800, Star Medical, Tokyo, Japan) and data acquired into a personal computer with the acquisition software (Eight Star, Star Medical, Tokyo, Japan). After confirmation of the incidence of interdigestive migrating contraction (IMC) at regular intervals, vehicle or a 5-HT4 receptor agonist was administered orally. Gut motility was then recorded for 8 h. Experiments in the postprandial state

Recording of motility was started as same as the experiment in the fasted state. After confirmation of IMC, 100 g /animal (cisapride and CJ-033466 studies) or 10 g/kg (mosapride and tegaserod studies) of solid meal was given to the dog. Two hours after feeding, vehicle or a 5-HT4 receptor agonist was administered orally. Gut motility was then recorded for 6 h. Antagonist study

SB-203186, a 5-HT4 receptor antagonist (10 mg/kg), or vehicle was subcutaneously administered one and a half hour after the fed. Thirty minutes later, CJ-033466 (0.1 mg/kg) was orally administered. Analysis of data

To quantify gastric motility, the areas of the contractions of the gastric antrum were determined by the processing software (Analyze II, Star Medical, Tokyo, Japan). In the fasted state study, the area surrounded by the contraction curve and the baseline for every 2 h period after administration was calculated. For standardization, the calculated areas were divided by the peak height of the last IMC before administration, and used as the motor index. In the postprandial state study, the area surrounded by the contraction curve and the baseline for every 1 h period after administration was calculated. For standardization, the calculated areas were divided by the area for 1 h period before administration, and expressed as a percentage. It was used as the motor index. Chemicals

CJ-033466, cisapride, mosapride, tegaserod and SB-203186 were synthesized at Pfizer Inc. Methylcellulose (MC) was purchased from Shin-Etsu Chemical Co. (Tokyo, Japan).

Cisapride, mosapride, tegaserod and CJ-033466 were suspended in 0.5% MC solution and administered orally at a volume of 5 mL. SB-203186 was dissolved in 0.9% saline at a volume of 0.2 mL/kg.

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Statistics Data were presented as mean ± S.E.M. Statistical analysis was performed with

Dunnett’s test using JMP software (SAS Institute Inc., Cary, NC, USA). P values of less than 0.05 were regarded as significant. 3. Results Effects of 5-HT4 receptor agonists on gastric motility under the fasted state in dogs.

Figure 2A shows the typical contractile patterns of the gastric antrum under fasted state in conscious dogs. The IMC was observed in intervals of approximately 90 min. Between the IMCs, few contractile activities were observed. Cisapride (0.3 – 3 mg/kg, p.o.) stimulated gastric motility in a dose-dependent manner (Fig. 3). The change in the motor index achieved statistical significance at the dose of 1 mg/kg during the 0 to 6 h period after administration. The stimulatory effect of cisapride 3 mg/kg lasted at least 8 h after the administration. Mosapride (0.3 – 3 mg/kg, p.o.) did not stimulate gastric motility (Fig. 4). Tegaserod (0.03 – 0.3 mg/kg, p.o.) dose-dependently stimulated gastric motility (Fig. 5). At the dose of 0.1 mg/kg, the stimulatory effect during the 0 to 2 h period after administration was statistically significant. CJ-033466 (0.01 – 0.3 mg/kg, p.o.) stimulated gastric motility in a dose-dependent manner (Fig. 6). The change in the motor index achieved statistical significance at the dose of 0.03 mg/kg during the 0 to 2 h period after administration. The stimulatory effect of CJ-033466 0.3 mg/kg lasted at least 8 h after administration. Effects of 5-HT4 receptor agonists on gastric motility under the postprandial state in dogs.

Figure 2C shows the typical contractile patterns of the gastric antrum under the postprandial state in conscious dogs. Approximately 8 h after feeding, the contractile pattern of postprandial state gradually changed to an irregular pattern, thereafter showed IMC-like strong contractions. Cisapride (0.3 – 3 mg/kg, p.o.) dose-dependently stimulated gastric motility (Fig. 7). At the dose of 1 mg/kg, the change in the motor index achieved statistical significance during the 0 to 4 h period. Mosapride (0.3 – 3 mg/kg, p.o.) stimulated gastric motility in a dose-dependent manner (Fig. 8). At the dose of 3 mg/kg, the stimulatory effect reached statistical significance during the 0 to 1 h and 2 to 4 h periods. Tegaserod (0.1 – 1 mg/kg, p.o.) stimulated gastric motility in a dose-dependent manner (Fig. 9). At the dose of 1 mg/kg, the stimulatory effect reached statistical significance during the 0 to 5 h period. CJ-033466 (0.01 – 0.3 mg/kg, p.o.)

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dose-dependently stimulated gastric motility (Fig. 10). At the dose of 0.03 mg/kg, the stimulatory effect was statistically significant during the 1 to 3 h period. The stimulatory effect of CJ-033466 reached maximum at the dose of 0.1 mg/kg, which lasted 5 h after administration. Effect of SB-203186 on CJ-033466-stimulated gastric motility in the postprandial state.

Pretreatment of SB-203186 (10 mg/kg, s.c.) completely blocked CJ-033466 (0.1 mg/kg, p.o.)-stimulated gastric motility (Fig. 11).

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CJ-033466 p.o.

Fasted state

Postprandial state

CJ-033466 p.o.

Vehicle p.o.

Feeding: Solid meal 100 g

2 hVehicle p.o.

(A)

(D)

(C)

(B)

1 h

200 g

CJ-033466 p.o.CJ-033466 p.o.

Fasted state

Postprandial state

CJ-033466 p.o.CJ-033466 p.o.

Vehicle p.o.Vehicle p.o.

Feeding: Solid meal 100 gFeeding: Solid meal 100 g

2 h2 hVehicle p.o.

(A)

(D)

(C)

(B)

1 h1 h

200 g200 g

Figure 2. Typical contractile patterns of the gastric antrum in conscious dogs. Effect of CJ-033466 (0.1 mg/kg, B, D) or vehicle (0.5% methylcellulose 5 mL/kg, A, C) on gastric motility under the fasted and postprandial states.

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Figure 3. Effect of cisapride on gastric motility under the fasted state in conscious dogs. Cisapride or vehicle (0.5% methylcellulose) was orally administrated. Changes in gastric motility were expressed by the motor index. The motor index was shown as relative AUC of the gastric motility for every 2 h period after the administration in which the peak height of the last interdigestive migrating contraction before the administration. Data are expressed as mean ± S.E.M. from 4 to 5 animals. *: P<0.05, **: P<0.01, compared with vehicle-treated group (Dunnett’s test).

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Vehicle 5 ml/head p.o. Mosapride 0.3 mg/kg p.o.


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Figure 4. Effect of mosapride on gastric motility under the fasted state in conscious dogs. Mosapride or vehicle (0.5% methylcellulose) was orally administrated. Changes in gastric motility were expressed by the motor index. The motor index was shown as relative AUC of the gastric motility for every 2 h period after the administration in which the peak height of the last interdigestive migrating contraction before the administration. Data are expressed as mean ± S.E.M. from 4 animals.

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Figure 5. Effect of tegaserod on gastric motility under the fasted state in conscious dogs. Tegaserod or vehicle (0.5% methylcellulose) was orally administrated. Changes in gastric motility were expressed by the motor index. The motor index was shown as relative AUC of the gastric motility for every 2 h period after the administration in which the peak height of the last interdigestive migrating contraction before the administration. Data are expressed as mean ± S.E.M. from 4 animals. *: P<0.05, **: P<0.01, compared with vehicle-treated group (Dunnett’s test).

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Figure 6. Effect of CJ-033466 on gastric motility under the fasted state in conscious dogs. CJ-033466 or vehicle (0.5% methylcellulose) was orally administrated. Changes in gastric motility were expressed by the motor index. The motor index was shown as relative AUC of the gastric motility for every 2 h period after the administration in which the peak height of the last interdigestive migrating contraction before the administration. Data are expressed as mean ± S.E.M. from 4 to 5 animals. *: P<0.05, **: P<0.01, compared with vehicle-treated group (Dunnett’s test).

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Time after administration (h)

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Figure 7. Effect of cisapride on gastric motility under the postprandial state in conscious dogs. After the confirmation of interdigestive migrating contraction, a solid meal was given the dog. Two hours after feeding, cisapride or vehicle (0.5% methylcellulose) was orally administrated. Changes in gastric motility were expressed by the percentage of motor index. The motor index was shown as relative AUC of the gastric motility for every 1 h period after the administration in which AUC of 1 h period before the administration was taken as 100%. Data are expressed as mean ± S.E.M. from 4 to 5 animals. *: P<0.05, **: P<0.01, compared with vehicle-treated group (Dunnett’s test).

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Figure 8. Effect of mosapride on gastric motility under the postprandial state in conscious dogs. After the confirmation of interdigestive migrating contraction, a solid meal was given the dog. Two hours after feeding, mosapride or vehicle (0.5% methylcellulose) was orally administrated. Changes in gastric motility were expressed by the percentage of motor index. The motor index was shown as relative AUC of the gastric motility for every 1 h period after the administration in which AUC of 1 h period before the administration was taken as 100%. Data are expressed as mean ± S.E.M. from 4 animals. *: P<0.05, compared with vehicle-treated group (Dunnett’s test).

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Figure 9. Effect of tegaserod on gastric motility under the postprandial state in conscious dogs. After the confirmation of interdigestive migrating contraction, a solid meal was given the dog. Two hours after feeding, tegaserod or vehicle (0.5% methylcellulose) was orally administrated. Changes in gastric motility were expressed by the percentage of motor index. The motor index was shown as relative AUC of the gastric motility for every 1 h period after the administration in which AUC of 1 h period before the administration was taken as 100%. Data are expressed as mean ± S.E.M. from 4 animals. *: P<0.05, **: P<0.01, compared with vehicle-treated group (Dunnett’s test).

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Figure 10. Effect of CJ-033466 on gastric motility under the postprandial state in conscious dogs. After the confirmation of interdigestive migrating contraction, a solid meal was given the dog. Two hours after feeding, CJ-033466 or vehicle (0.5% methylcellulose) was orally administrated. Changes in gastric motility were expressed by the percentage of motor index. The motor index was shown as relative AUC of the gastric motility for every 1 h period after the administration in which AUC of 1 h period before the administration was taken as 100%. Data are expressed as mean ± S.E.M. from 4 to 5 animals. *: P<0.05, **: P<0.01, compared with vehicle-treated group (Dunnett’s test).

- 19 -

Feeding

Feeding

Vehicles.c.

SB-203186s.c.

CJ-033466 p.o.

CJ-033466 p.o.

1 h

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(A)

(B)

Feeding

Feeding

Vehicles.c.

SB-203186s.c.

CJ-033466 p.o.

CJ-033466 p.o.

1 h

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Feeding

Feeding

Vehicles.c.

SB-203186s.c.

CJ-033466 p.o.

CJ-033466 p.o.

1 h

100 g

(A)

(B)

Figure 11. Effect of SB-203186 on CJ-033466-stimulated gastric motility under the postprandial state in conscious dogs. After the confirmation of interdigestive migrating contraction, a solid meal was given the dog. (A) Vehicle (0.9% saline 0.2 mL/kg) or (B) SB-203186 (10 mg/kg) was subcutaneously administered 1.5 h after feeding. Thirty minutes later, CJ-033466 (0.1 mg/kg) was orally administrated.

- 20 -

4. Discussion In this chapter, cisapride, tegaserod and CJ-033466 dose-dependently stimulated gastric motility under both fasted and postprandial states in conscious dogs. Mosapride only stimulated gastric motility under the postprandial state. Potencies to stimulatory effect on gastric motility are CJ-033466 > tegaserod > cisapride in the fasted state and CJ-033466 > tegaserod = cisapride > mosapride in the postprandial state. The stimulatory effect of CJ-033466 was completely blocked by a 5-HT4 receptor antagonist, SB-203186. This result indicates that CJ-033466 stimulate gastric motility via 5-HT4 receptors. The enhancement of gastric motility by cisapride under both fasted and postprandial states in conscious dogs is well reported (49-52). In these reports, cisapride was administrated intravenously and marked stimulatory effect was observed at doses above 0.1 mg/kg. Tanaka et al. (53) reported that intraduodenal administration of cisapride significantly enhanced gastric motility under the postprandial state in conscious dogs at the dose of 1 mg/kg. This potency is consistent with the present results using oral administration. Pretreatment of atropine or hexamethonium in the fasted state, or GR113808 (5-HT4 receptor antagonist) in the postprandial state completely inhibited gastroprokinetic effect of cisapride (49, 51, 52), suggesting the involvement of preganglionic cholinergic neurons and 5-HT4 receptors in the stimulatory effect of cisapride. The effect of tegaserod on the upper gastrointestinal tract has not been intensively studied. In the present study, tegaserod demonstrated apparent stimulatory effect on gastric motility which is more potent than that of cisapride. The stimulatory effect of tegaserod on gastric motility was statistically significant at 0.1 and 1 mg/kg, p.o. in the fasted and postprandial states, respectively. When tegaserod was taken with food, the absorption was slowed and the AUC value was reduced approximately 2-fold in humans (54, 55). This food effects may influence on the potency of tegaserod under the postprandial state. The stimulatory effect of mosapride on gastric motility was only observed under the postprandial state. Consistent with this result, gastroprokinetic effect of mosapride under the postprandial state was reported with intravenous or intraduodenal administration (51, 56). They reported that the stimulatory effect of mosapride was blocked by atropine or GR113808, but the stimulatory effect was observed in vagally denervated pouch. These results indicated the involvement of the postganglionic cholinergic neurons and 5-HT4 receptors in the stimulatory effect of mosapride. Yoshida

- 21 -

et al. (56) suggested that different neurological pathway involved with cisapride and mosapride is due to their different pharmacological profile. This discrepancy may also depend on the gastric motility induced under different feeding conditions, fasted or fed. Mosapride did not stimulate gastric motility under the fasted state. The functional activity of mosapride on the isolated rat TMM is weak in in-house data (Fig. 1). Mine et al. (51) also reported that EC50 values of mosapride and cisapride on rat TMM is 208 and 39 nM, respectively. In addition, mosapride enhanced the electrically evoked contractions of guinea pig ileum and evoked the contractions of guinea pig distal colon with EC50 values of 73 and 3029 nM, respectively. Those of EC50 values of cisapride are 48 and 32 nM, respectively (51). These results indicate that mosapride may need a pre-stimulated condition to demonstrate its activity. Therefore, a dose higher than 3 mg/kg may be needed to stimulate the gastric motility under fasted condition. Another reason for the inactivity of mosapride under fasted state is the presence of a metabolite of mosapride. It is reported that the major metabolite of mosapride has potent 5-HT3 receptor antagonistic activity (38). 5-HT3 receptor antagonists inhibited the IMC in the fasted state and did not influence gastric motility in the postprandial state (57). Therefore, 5-HT3 antagonism of the metabolite may block the stimulatory effect of mosapride in the fasted state.

- 22 -

Chapter II: Effects of 5-HT4 receptor agonists on gastric emptying and gastric motility in rats 1. Introduction

5-HT4 receptor agonists demonstrate the enhancement of canine gastric motility, except for mosapride in the fasted state, in Chapter I. Although most of the time enhancement of gastric motility correlates the acceleration of gastric emptying, the number and/or force of contractions are increased does not mean that empting is enhanced. Antropyloroduodenal incoordination or retrograde peristalsis may impair gastric emptying. It is important to understand the correlation between gastric motility and gastric emptying. Thus, in this chapter, both rat gastric emptying and gastric motility models are used to investigate the efficacy of 5-HT4 receptor agonists.

The isolated rat TMM is generally used for examining the 5-HT4 receptor agonist activity (36, 37). Not only in-house data, but also cisapride, mosapride and tegaserod data are reported in this functional assay (39, 51). In the present experiment, comparison of in vitro and in vivo data from rats was performed, in order to clarify the properties of 5-HT4 receptor agonists.

The blockade of 5-HT3 receptor inhibits gastric motility under the fasted state in dogs (57) and humans (58). In contrast, the blockade of 5-HT3 receptor accelerates gastric emptying in rats (59-61). Unlikely, mosapride did not stimulate canine gastric motility under the fasted state. One of the reasons for the lack of stimulatory effect of mosapride may be the 5-HT3 antagonism of its metabolite. The result from rat gastric emptying study using 5-HT4 receptor antagonist may clarify the reason.

In this chapter, effects of cisapride, mosapride, tegaserod and CJ-033466 on gastric emptying and gastric antral motility in free moving conscious rats were examined and the correlation between them was evaluated. In addition, effects of ondansetron (5-HT3 receptor antagonist) and α-methyl-5-HT (5-HT2 receptor agonist) were examined to understand the results from cisapride, mosapride, tegaserod and CJ-033466. 2. Materials and Methods Animals

Male Crj:SD(IGS) rats weighing 185-255 g (Charles River Japan, Inc., Shiga, Japan) were used. The animals were maintained on ordinary laboratory chow and tap water ad libitum under a constant 12-hr light-dark cycle.

- 23 -

Gastric emptying in rats Rats were fasted overnight with free access to water. Water was deprived 2 hr before

starting the experiment. A semi-solid meal (methylcellulose: 7 g, glucose: 7.5 g, cornstarch: 9 g, casein: 15 g in 275 ml of water) 3 mL/head was given orally. Thirty minutes later, rats were euthanized with CO2. The stomach was exposed through laparotomy, quickly ligated at both pylorus and cardia, then removed and weighed. The stomach was opened and rinsed with saline, and weighed again. Gastric emptying rate was calculated the following formula. Gastric emptying rate (%) = (1-Weight of meal left in the stomach / Weight of given meal) x 100. Cisapride, mosapride, tegaserod ondansetron or vehicle was orally administered to rats 15 min before the semi-solid meal administration. CJ-033466 or vehicle was orally administered to rats 5 min before the semi-solid meal administration. Alpha-methyl-5-HT or vehicle was intravenously administered to rats 10 min before the semi-solid meal administration.

In order to evaluate the effect of SB-203186 on gastrokinetic effects of 5-HT4 agonists, SB-203186 (10 mg/kg), or vehicle was subcutaneously administered to rats 5 min before the 5-HT4 receptor agonist administration. Gastric motility in rats

Rats were fasted overnight with free access to water before an abdominal operation. Animals were anaesthetized with ketamine 50 mg/xylazin 20 mg/kg i.m. and motility recording device was implanted as follows. A manometric catheter and a catheter for intragastric administration (Intravenous catheter: 3Fr, 1 mm diameter, ATOM, Tokyo, Japan) were inserted through the fistula in the gastric body and the tips placed at the gastric antrum. Catheters were fixed to the gastric wall by sutures, which ran subcutaneously to emerge at the top of the neck and were secured at the animal’s skin. A catheter (Indwelling feeding tube: 3Fr, 1 mm diameter, ATOM, Tokyo, Japan) was also placed in the left femoral vein. This ran subcutaneously to emerge at the top of the neck beside the manometric catheter, and used for i.v. administration. Animals were allowed to recover for 1 week before the measurement of gastric motility.

For measurement of gastric antal motility, rats were fasted overnight with free access to water. Gastric antral motility was measured in conscious, freely moving rats by a manometric method. On the day of the experiment, the manometric catheter was connected to a pressure transducer (DX-100: Nihon Koden, Tokyo, Japan) and continuously infused with distilled water at a rate of 3 mL/h by a low-compliance capillary infusion system using pressure cuff. The data was recorded and stored in a PowerLab system (ADInstruments Pty Ltd, Castle Hill, Australia). After recording the

- 24 -

basal gastric motility for at least 30 min, SB-203186 or vehicle was administered intravenously. Five minutes later, cisapride, mosapride, tegaserod or CJ-033466 was administered into the stomach. Then gastric motility was continuously measured for 60 min. For ondansetron, the protocol was the same as the 5-HT4 agonist study, except for without i.v. administration.

To quantify gastric motility, the areas of the contractions of the gastric antrum were determined by the PowerLab system. The area surrounded by the contraction curve and the baseline for every 5 min period after the first administration (i.v. or i.g.) was calculated. For standardization, on the period before the first drug administration, the average of the 15 min area was calculated and expressed as 5 min period. It was used as the motor index. Chemicals

Cisapride, mosapride, tegaserod, CJ-033466, ondansetron and SB-203186 were synthesized at Pfizer Inc. Alpha-methyl-5-HT maleate was purchased from Sigma (St. Louis, MO, USA). Casein was purchased from Sigma-Aldrich Japan (Tokyo, Japan). Methylcellulose (for test meal), D(+)-glucose and cornstarch were purchased from Wako pure chemical (Osaka, Japan). Methylcellulose (MC) for vehicle was purchased from Shin-Etsu Chemical Co. (Tokyo, Japan).

Cisapride, mosapride, tegaserod, CJ-033466 and ondansetron were suspended with 0.5% MC in water at a volume of 2 mL/kg. SB-203186 and α-methyl-5-HT were solved with 0.9% saline at a volume of 2 mL/kg. Statistics

Data were presented as mean ± S.E.M. For the gastric emptying dose-response study, the comparison between the control group and the treated group was carried out by one-way analysis of variance (ANOVA) with Dunnett’s test. For the gastric emptying antagonist study, the comparison between the control group and the antagonist-treated group was carried out by unpaired t-test with Welch's correction. P values of less than 0.05 were regarded as significant. 3. Results Effects of 5-HT4 receptor agonists on gastric emptying.

Cisapride (0.1 – 3 mg/kg, p.o.) accelerated gastric emptying in a dose-dependent manner (Fig. 12). Acceleratory effects of cisapride were statistically significant at doses

- 25 -

of 1 and 3 mg/kg. Mosapride (0.1 – 3 mg/kg, p.o.) dose-dependently accelerated gastric emptying (Fig. 13). At the dose of 0.3 mg/kg, stimulatory effect of mosapride reached statistically significant. Maximal effect was observed at 1 mg/kg. On the other hand, tegaserod (0.1 – 3 mg/kg, p.o.) did not accelerate gastric emptying (Fig. 14). CJ-033466 accelerated gastric emptying in a dose-dependent manner (Fig. 15). At the dose of 3 mg/kg, stimulatory effect of CJ-033466 was statistically significant. Effects of SB-203186 on 5-HT4 receptor agonists-accelerated gastric emptying.

Pretreatment with SB-203186 (1 – 10 mg/kg, s.c.) inhibited cisapride (3 mg/kg, p.o.)-accelerated gastric emptying (Fig. 16). At the dose of 10 mg/kg, SB-203186 completely inhibited the stimulatory effect of cisapride. Therefore the following antagonist study, 10 mg/kg of SB-203186 was pretreated. SB-203186 inhibited both mosapride (1 mg/kg, p.o.) and CJ-033466 (3 mg/kg, p.o.)-accelerated gastric emptying (Figs. 17, 18). SB-203186 delayed gastric emptying treated with tegaserod (3 mg/kg, p.o.), but was not statistically significant (Fig. 17). Effects of ondansetron and α-methyl-5-HT on gastric emptying. Ondansetron (0.1 – 3 mg/kg, p.o.) accelerated gastric emptying significantly (Fig. 19). At the dose of 0.1 mg/kg, the stimulatory effect already reached the maximum. Alpha-methyl-5-HT (0.01 – 1 mg/kg, i.v.) did not accelerate gastric emptying (Fig. 20). Effects of 5-HT4 receptor agonists on gastric motility.

Cisapride (3 mg/kg, i.g.) markedly stimulated gastric motility (Fig. 21). Mosapride (1 mg/kg, i.g.) stimulated gastric motility (Fig. 22), but tegaserod (3 mg/kg, i.g.) did not show any effect on gastric motility (Fig. 23). CJ-033466 (3 mg/kg, i.g.) stimulated gastric motility (Fig. 24). The stimulatory effect of CJ-033466 was short duration of action. Effects of SB-203186 on 5-HT4 receptor agonists-stimulated gastric motility.

SB-203186 (10 mg/kg, i.v.) slightly increased gastric motility at 30 to 40 min after the administration. Pretreatment with SB-203186 (10 mg/kg, i.v.) completely inhibited cisapride (3 mg/kg, p.o.), mosapride (1 mg/kg, p.o.) or CJ-033466 (3 mg/kg, p.o.)-stimulated gastric motility (Figs. 21 - 24). Effects of ondansetron and α-methyl-5-HT on gastric motility. Ondansetron (1 mg/kg, p.o.) did not influence gastric motility (Fig. 25). Preliminary

- 26 -

data, α-methyl-5-HT (0.3mg/kg, i.v.) increased gastric motility (Fig. 26, n = 1).

- 27 -

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Figure 12. Effect of cisapride on gastric emptying in conscious rats. Cisapride orvehicle was orally administered to rats 15 min before test meal administration.Data are expressed as mean ± S.E.M. from 6 to 8 animals. **: P<0.01,compared with vehicle-treated group (One-way ANOVA followed by theDunnett’s test).

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Figure 13. Effect of mosapride on gastric emptying in conscious rats. Mosaprideor vehicle was orally administered to rats 15 min before test meal administration.Data are expressed as mean ± S.E.M. from 6 to 8 animals. **: P<0.01,compared with vehicle-treated group (One-way ANOVA followed by theDunnett’s test).

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Figure 14. Effect of tegaserod on gastric emptying in conscious rats. Tegaserod orvehicle was orally administered to rats 15 min before test meal administration.Data are expressed as mean ± S.E.M. from 6 to 7 animals.

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Figure 15. Effect of CJ-033466 on gastric emptying in conscious rats. CJ-033466or vehicle was orally administered to rats 5 min before test meal administration.Data are expressed as mean ± S.E.M. from 7 to 8 animals. **: P<0.01,compared with vehicle-treated group (One-way ANOVA followed by theDunnett’s test).

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Figure 16. Effect of SB-203186 on the gastrokinetic response to cisapride inconscious rats. SB-203186 or 0.9% saline was subcutaneously injected to rats 20 min before test meal administration. Cisapride or 0.5% methylcellulose (MC) was orally administered to rats 15 min before test meal administration. Data are expressed as mean ± S.E.M. from 5 to 7 animals. ##: P<0.01, compared with saline s.c. and 0.5% MC p.o.-treated group (Unpaired t-test with Welch’s correction). *: P<0.05, **: P<0.01, compared with 0.9% saline s.c and cisapride p.o.-treated group (One-way ANOVA followed by the Dunnett’s test).

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Figure 17. Effect of SB-203186 on the gastrokinetic response to cisapride, tegaserodor mosapride in conscious rats. SB-203186 or 0.9% saline was subcutaneouslyinjected to rats 20 min before test meal administration. Cisapride, tegaserod, mosapride or 0.5% methylcellulose (MC) was orally administered to rats 15 minbefore test meal administration. Data are expressed as mean ± S.E.M. from 5 to 7 animals. ##: P<0.01, compared with 0.9% saline s.c. and 0.5% MC p.o.-treated group. **: P<0.01, compared with saline s.c and cisapride or mosapride p.o.-treated group (Unpaired t-test with Welch’s correction).

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Figure 18. Effect of SB-203186 on the gastrokinetic response to CJ-033466 in conscious rats. SB-203186 or 0.9% saline was subcutaneously injected to rats 10 min before test meal administration. CJ-033466 or 0.5% methylcellulose (MC) was orally administered to rats 5 min before test meal administration. Data are expressed as mean ± S.E.M. from 7 to 8 animals. ##: P<0.01, compared with 0.9% saline s.c. and 0.5% MC p.o.-treated group. **: P<0.01, compared with saline s.c and CJ-033466 p.o.-treated group (Unpaired t-test with Welch’s correction).

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Figure 19. Effect of ondansetron on gastric emptying in conscious rats. Ondansetron or vehicle was orally administered to rats 15 min before test meal administration.Data are expressed as mean ± S.E.M. from 6 to 8 animals. *: P<0.05, **: P<0.01, compared with vehicle-treated group (One-way ANOVA followed by the Dunnett’s test).

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α-methyl-5HT (mg/kg, i.v.)

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Figure 20. Effect of α-methyl-5HT on gastric emptying in conscious rats. α-methyl-5HT or vehicle was intravenously injected to rats 10 min before test meal administration. Data are expressed as mean ± S.E.M. from 6 to 8 animals.

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Figure 21. Effect of cisapride on gastric motility in conscious rats. Cisapride (3 mg/kg) or vehicle (0.5% methylcellulose 2 mL/kg) was intragastricaly administered at 0 min. Five minutes before the administration, SB-203186 (10 mg/kg) or 0.9% saline (2 mL/kg) was intravenously administered. Data are expressed as mean ± S.E.M. from 3 to 4 animals.

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Saline iv + MC igSaline iv + MC igSB-203186 iv + MC igSB-203186 iv + MC igSaline iv + Mosapride igSaline iv + Mosapride igSB-203186 iv + Mosapride igSB-203186 iv + Mosapride ig

Figure 22. Effect of mosapride on gastric motility in conscious rats. Mosapride (1 mg/kg) or vehicle (0.5% methylcellulose 2 mL/kg) was intragastricaly administered at 0 min. Five minutes before the administration, SB-203186 (10 mg/kg) or 0.9% saline (2 mL/kg) was intravenously administered. Data are expressed as mean ± S.E.M. from 3 to 4 animals.

- 38 -

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Saline iv + MC igSaline iv + MC igSB-203186 iv + MC igSB-203186 iv + MC igSaline iv + Tegaserod igSaline iv + Tegaserod igSB-203186 iv + Tegaserod igSB-203186 iv + Tegaserod ig

Figure 23. Effect of tegaserod on gastric motility in conscious rats. Tegaserod (3 mg/kg) or vehicle (0.5% methylcellulose 2 mL/kg) was intragastricaly administered at 0 min. Five minutes before the administration, SB-203186 (10 mg/kg) or 0.9% saline (2 mL/kg) was intravenously administered. Data are expressed as mean ± S.E.M. from 3 to 4 animals.

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Figure 24. Effect of CJ-033466 on gastric motility in conscious rats. CJ-033466 (3 mg/kg) or vehicle (0.5% methylcellulose 2 mL/kg) was intragastricaly administered at 0 min. Five minutes before the administration, SB-203186 (10 mg/kg) or 0.9% saline (2 mL/kg) was intravenously administered. Data are expressed as mean ± S.E.M. from 3 to 4 animals.

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Figure 25. Effect of ondansetron on gastric motility in conscious rats. Ondansetron (1 mg/kg) or vehicle (0.5% methylcellulose 2 mL/kg) was intragastricaly administered at 0 min. Five minutes before the administration, 0.9% saline (2 mL/kg) was intravenously administered for control group. Data are expressed asmean ± S.E.M. from 3 to 4 animals.

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50 mmHg

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50 mmHg50 mmHg

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α-methyl-5-HT 0.3 mg/kg, i.v.α-methyl-5-HT 0.3 mg/kg, i.v.

Figure 26. Effect of α-methyl-5HT on gastric motiltiy in conscious rats. α-methyl-5HT was intravenously administered.

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4. Discussion In this chapter, cisapride, mosapride and CJ-033466 dose-dependently accelerated gastric emptying in conscious rats. However, tegaserod did not accelerate gastric emptying. Potencies to stimulatory effect on gastric emptying are mosapride > cisapride > CJ-033466. Consistent with this result, oral administration of mosapride demonstrated comparable or slight potent acceleratory effect than cisapride on gastric emptying in normal rats (62, 63). In these report, at a dose of 1 mg/kg of mosapride and cisapride reached statistical significant. With intravenous administration, cisapride-accelerated gastric emptying was observed approximately 10 times less dose than that of mosapride (61). This result consists with in vitro potency (51). Both cisapride and mosapride stimulated gastric antral motility. These results correlated with acceleratory effects on gastric emptying. On the other hand, CJ-033466 accelerated gastric emptying significantly, but the potency is 3 times less than that of cisapride. CJ-033466 demonstrated the potent but short-lasting stimulatory effect on gastric antral motility. This short duration of action may result in the weak potency of CJ-033466 in rats. It is reported that mosapride restored cisplatin-induced delayed gastric emptying (63). This effect was stronger than that of cisapride. Cisplatin is an antineoplastic agent, which induce side effects of nausea and emesis in clinical due to activation of 5-HT3 receptors located in the gastrointestinal tract and/or central nervous system (64, 65). A metabolite of mosapride has 5-HT3 antagonism which potency is estimated to be 20-fold less than a 5-HT3 antagonist, ondansetron (38, 66). However, in the present study, pretreatment with SB-203186, a 5-HT4 receptor antagonist, completely blocked mosapride-stimulated gastric emptying and gastric antral motility, indicating that stimulatory effect of mosapride mainly induced via 5-HT4 receptors. From these results, it may suggest that mosapride did not stimulate canine gastric motility under the fasted state due to low activity on basal condition. SB-203186 also completely blocked cisapride- and CJ-033466-accelerated gastric emptying and gastric antral motility. The stimulatory effects of cisapride and CJ-033466 may be induced via 5-HT4 receptors. Tegaserod stimulated neither gastric emptying nor gastric antral motility in conscious rats. SB-203186 delayed tegaserod-treated gastric emptying, suggesting that tegaserod accelerated gastric emptying in some part via 5-HT4 receptors. Other than 5-HT4 agonism, tegaserod may have pharmacological mechanism to delay gastric emptying. Recent study has revealed that tegaserod has a potent 5-HT2B antagonism which is comparable to 5-HT4 agonism (39). The 5-HT2B receptor was first discovered on rat

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fundic longitudinal muscles and has been well characterized (67-69). 5-HT-induced fundic contractions were blocked by 5-HT2B receptor antagonists. Therefore, tegaserod may retain the administrated meal in the fundus via 5-HT2B antagonism. In order to confirm this hypothesis, effect of α-methyl-5-HT, a 5-HT2 agonist on gastric emptying was examined. Although a systemic side effect was observed in rats at the dose of 1 mg/kg, i.v. of α-methyl-5-HT, up to this dose, α-methyl-5-HT did not accelerate gastric emptying. At the dose of 0.3 mg/kg, α-methyl-5-HT stimulated gastric antral motility in the preliminary data. Consistent with this result, Beattie et al. (39) also reported α-methyl-5-HT-induced fundic contractions in anesthetized rats. Antropyloroduodenal incoordination like pylorus sphincter contraction may occur. However, further studies are needed to clarify why α-methyl-5-HT-stimulated gastric motility did not accelerate gastric emptying in rats. On the other hand, ondansetron enhanced gastric emptying significantly, but did not stimulate gastric antral motility. Not only ondansetron but also other 5-HT3 antagonists are known to increase the rate of gastric emptying in rats (59-61). Pretreatment with atropine or tetrodotoxin (TTX) abolished these gastric emptying accelerations, indicating the participation of cholinergic neurons (59). Generally, the stimulation of cholinergic neurons induces enhancement of gastric motility. Ondansetron may stimulate other regions of gastric motility to accelerate gastric emptying.

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Chapter III: Pharmacological characterization of 5-HT-receptor subtypes in circular muscle from the rat stomach 1. Introduction

It is generally accepted that gastric emptying is regulated by the coordination of the proximal and distal stomach motilities. 5-HT has proven to be an important mediator of gastrointestinal motility (70, 71). The stimulation of 5-HT1B/1D receptors induces gastric accommodation in humans (22) and dogs (72) and delays gastric emptying in humans (73, 74). The blockade of 5-HT3 receptors inhibits gastrointestinal motility in humans (58). In addition, the stimulation of 5-HT4 receptors elicits gastrointestinal contractile activity and increases the rate of gastric emptying in humans (75, 76) and dogs (50, 77, 78, Chapter I). With regard to gastric smooth muscle activity, it is generally accepted that the 5-HT1B/1D and 5-HT7 receptors are relaxant whereas the 5-HT2, 5-HT3, and 5-HT4 receptors are contractile. Although the gastric contractility of 5-HT has been reported in a variety of species and stomach regions, regional and functional differences of 5-HT-receptor subtypes have been fully investigated only in the guinea pig stomach (79-82). In the guinea pig stomach, the fundus presents the relaxant 5-HT1 receptors and the contractile 5-HT2 and 5-HT3 receptors, the corpus presents the relaxant 5-HT1 receptors and the contractile 5-HT3 and 5-HT4 receptors, and the antrum presents the contractile 5-HT2, 5-HT3 and 5-HT4 receptors. However, the in vivo studies for examining gastric motility and gastric emptying using guinea pigs are limited. On the other hand, the rat is widely used as in vivo models to study gastric motility (39, 83, 84) and gastric emptying rate (59, 62, 85). The stimulation of 5-HT2 receptors increases gastric motility (39), the blockade of 5-HT3 receptors accelerates gastric emptying (59), and the stimulation of 5-HT4 receptors increases gastric motility (83) and accelerates gastric emptying (59, 62). These findings are also reported in Chapter II. In in vitro, pharmacological characterization of the contractile response to 5-HT in the rat stomach regions still remains unclear, except for the contractile 5-HT2B receptors in longitudinal muscle strips of the fundus (67-69). This chapter was, therefore, designed to provide further information regarding functions of 5-HT receptor subtypes in circular muscle strips from the antrum, corpus and fundus of the rat stomach. 2. Materials and Methods Preparation of smooth muscle strips from the rat stomach

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Male Crj:SD (IGS) rats (Charles River Japan, Inc., Shiga, Japan) weighing 300-400 g were euthanized with Isoflurane inhalation. The stomach was excised and immediately cut into strips of fundus, corpus and antrum, in a circular fashion. The mucosa was removed from the tissue and muscle layers. Measurements of mechanical activity

The strips of fundus, corpus or antrum were placed into a 10-mL organ bath containing Krebs-Ringer solution of the following composition: 118 mM NaCl, 4.8 mM KCl, 2.5 mM CaCl2, 1.19 mM MgSO4, 25.0 mM NaHCO3, 1.18 mM KH2PO4, 11 mM glucose and 0.12 mM ascorbic acid, oxygenated with 95% O2 and 5% CO2 and maintained at 37℃. Approximately 1 g of resting tension was applied, and the tension was kept constant by re-adjustment during the 60-min equilibration period. Mechanical responses were recorded by means of an isometric transducer (TB-612T; Nihon Kohden, Tokyo, Japan). Single doses of agonists were applied and then washed out on an interval of 30 to 35 min. To evaluate the effects of antagonists, the tissue strips were exposed to the antagonists 10 min before and during application of agonists; the applied dose of each antagonist was determined according to our preliminary trials or the doses used in the literature. The concentration-response curves of agonists were performed in concentrations from 3 x 10-9 to 3 x 10-6 M for the fundic strip and from 3 x 10-7 to 3 x 10-4 M for the corporal and antral strips. Amplitudes of contraction and relaxation are represented relative to amplitude of contraction elicited by 10-5 M carbachol. Chemicals

Carbachol (CCh), atropine sulfate monohydrate and tetrodotoxin (TTX) were purchased from Wako Pure Chemicals (Osaka, Japan). 5-Hydroxytryptamine hydrochloride (5-HT), Nω-Nitro-L-arginine (L-NOARG, NO synthetase inhibitor), methysergide maleate, ketanserin tartrate and SB-269970 (selective 5-HT7 receptor antagonist) were purchased from Sigma (St. Louis, MO, U.S.A.). WAY-100635 maleate (selective 5-HT1A receptor antagonist), GR127935 hydrochloride (5-HT1B/1D receptor antagonist) and cinanserin hydrochloride (selective 5-HT2 receptor antagonist) were purchased from Tocris Cookson (Ellisville, MO, U.S.A.). RS-127445 (selective 5-HT2B receptor antagonist), ondansetron (selective 5-HT3 receptor antagonist) and SB-203186 (selective 5-HT4 receptor antagonist) were synthesized at Pfizer Inc. All the drugs, except for L-NOARG, RS-127445 and ondansetron, were dissolved in distilled water to produce 10-2 or 10-3 M and then diluted to the required concentrations with Krebs-Ringer solution. L-NOARG was dissolved in Krebs-Ringer solution to produce

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10-2 M. RS-127445 was dissolved in dimethyl sulfoxide to produce 10-3 M and then diluted to the required concentrations with distilled water. Ondansetron was dissolved in 40% sulfobutylether-β-cyclodextrin to produce 10-2 M and then diluted to the required concentrations with distilled water. Statistics

Data were presented as mean ± S.E.M. The comparison between the control group and the antagonist-treated group was carried out by unpaired t-test with Welch's correction. P values of less than 0.05 were regarded as significant. 3. Results Effect of exogenous 5-HT on the antral, corporal and fundic circular muscle.

The strips from gastric antrum and corpus displayed spontaneous phasic and cyclic activity, respectively. Conversely, the rat gastric fundic circular muscle strips did not show spontaneous activity. 5-HT evoked both tonic and phasic contractile activity in gastric antral strips and tonic contractile activities in gastric corporal and fundic strips (Fig. 27). 5-HT evoked concentration-dependent contraction of strips from antrum (5-HT at 10-8 to 3 x 10-4 M), corpus (5-HT at 10-8 to 3 x 10-4 M) and fundus (5-HT at 10-10 to 3 x 10-6 M) (Fig. 28). EC50 values for 5-HT were 1.97 x 10-5 M in the antrum, 8.22 x 10-6 M in the corpus and 4.64 x 10-8 M in the fundus. The contractile responses of the antral strips to 5-HT were potentiated by pretreatment with tetrodotoxin (TTX, 10-6 M) and tended to be inhibited by pretreatment with atropine (10-6 M). However the inhibitory effect of atropine was not statistically significant. The contractions of the corporal and fundic strips to 5-HT were not affected by pretreatment with either TTX (10-6 M) or atropine (10-6 M) (Figs. 29, 30). The contractile responses of the antral strips to 5-HT were potentiated by pretreatment with Nω-Nitro-L-arginine (L-NOARG, 10-4 M) (Fig. 31). Effects of 5-HT receptor antagonists on the 5-HT-induced contractions of the antral circular muscle.

Pretreatment with WAY-100635 (10-6 M), GR127935 (10-7 M), ketanserin (10-7 and 10-6 M), RS-127445 (10-7 M), ondansetron (10-6 M) and SB-269970 (10-6 M) had no effect on the 5-HT-induced contractions of the antral strips (Fig. 32A, B, D, E, F and H). Pretreatment with methysergide (10-7 M) inhibited the 5-HT-induced contractions of the antral strips (Fig. 32C). The inhibitory effect of ketanserin was not significant, but at

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10-6 M ketanserin tended to attenuate the contractile responses (Fig. 32D). Pretreatment with SB-203186 (10-6 M) enhanced the 5-HT-induced contractions of the antral strips (Fig. 32G). Effects of 5-HT receptor antagonists on the 5-HT-induced contractions of the corporal circular muscle.

Pretreatment with WAY-100635 (10-6 M), GR127935 (10-7 M), RS-127445 (10-7 M), ondansetron (10-6 M), SB-203186 (10-6 M) and SB-269970 (10-6 M) had no effect on the 5-HT-induced contractions of the corporal strips (Fig. 33A, B, E, F, G and H). Pretreatment with methysergide (10-7 M) and ketanserin (10-7 and 10-6 M) inhibited the 5-HT-induced contractions of the corporal strips (Fig. 33C and D). Effects of 5-HT receptor antagonists on the 5-HT-induced contractions of the fundic circular muscle.

Pretreatment with WAY-100635 (10-6 M), GR127935 (10-7 M), ketanserin (10-7 and 10-6 M), ondansetron (10-6 M) and SB-269970 (10-6 M) had no effect on the 5-HT-induced contractions of the fundic strips (Fig. 34A, B, D, F and H). The effect of ketanserin at 10-7 M was not significant, but showed a tendency to enhance the contractile response to 5-HT (Fig. 34D). Pretreatment with cinanserin (10-6 M) enhanced the 5-HT-induced contractions of the fundic strips (Fig. 35). Pretreatment with SB-203186 (10-6 M) also enhanced the 5-HT-induced contractions of the fundic strips (Fig. 34G). Pretreatment with methysergide (10-7 M) and RS-127445 (10-7 M) inhibited the 5-HT-induced contractions of the fundic strips (Fig. 34C and E).

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Figure 27. Trace showing typical effect of 5-HT on the strips from antrum (A), corpus (B), and fundus (C) of the rat stomach.

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Figure 28. Concentration-response curves for 5-hydroxytryptamine (5-HT) of thestrips from antrum (A), corpus (B), and fundus (C) of the rat stomach. Thecontractions induced by 5-HT was represented as their amplitude relative toamplitude of the contraction induced by 10-5 M carbachol. Each point represents the mean ± S.E.M. from 5 animals.

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Figure 29. Effects of atropine on 5-HT-induced contractions of the strips from antrum (A), corpus (B), and fundus (C) of the rat stomach. Atropine was applied 10 min before addition of 5-HT. The contractions induced by 5-HT was represented as their amplitude relative to amplitude of the contraction induced by 10-5 M carbachol.Each point represents the mean ± S.E.M. from 7-10 animals.

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Tetrodotoxin 10-6 MControl

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Figure 30. Effects of tetrodotoxin (TTX) on 5-HT-induced contractions of the strips from antrum (A), corpus (B), and fundus (C) of the rat stomach. TTX was applied 10min before addition of 5-HT. The contractions induced by 5-HT was represented as their amplitude relative to amplitude of the contraction induced by 10-5 M carbachol.Each point represents the mean ± S.E.M. from 7-10 animals. *: P<0.05, **: P<0.01, compared with the value in the absence of TTX (unpaired t-test with Welch’s correction).

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Figure 31. Effect of Nω-Nitro-L-arginine (L-NOARG) on 5-HT-inducedcontractions of the strips from corpus of the rat stomach. L-NOARG wasapplied 10 min before addition of 5-HT. The contractions induced by 5-HTwas represented as their amplitude relative to amplitude of the contractioninduced by 10-5 M carbachol. Each point represents the mean ± S.E.M.from 6 animals. **: P<0.01, compared with the value in the absence ofantagonists (unpaired t-test with Welch’s correction).

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ControlSB-203186 10-6 MControl

Figure 32. Effects of 5-HT receptor antagonists on 5-HT-induced contractions of thestrips from antrum of the rat stomach. WAY-100635 (A), GR127935 (B),methysergide (C), ketanserin (D), RS-127445 (E), ondansetron (F), SB-203186 (G)or SB-269970 (H) was applied 10 min before addition of 5-HT. The contractionsinduced by 5-HT was represented as their amplitude relative to amplitude of thecontraction induced by 10-5 M carbachol. Each point represents the mean ± S.E.M.from 7-9 animals. *: P<0.05, **: P<0.01, compared with the value in the absence ofantagonists (unpaired t-test with Welch’s correction).

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Figure 33. Effects of 5-HT receptor antagonists on 5-HT-induced contractions of thestrips from corpus of the rat stomach. WAY-100635 (A), GR127935 (B),methysergide (C), ketanserin (D), RS-127445 (E), ondansetron (F), SB-203186 (G)or SB-269970 (H) was applied 10 min before addition of 5-HT. The contractionsinduced by 5-HT was represented as their amplitude relative to amplitude of thecontraction induced by 10-5 M carbachol. Each point represents the mean ± S.E.M.from 7-9 animals. *: P<0.05, **: P<0.01, compared with the value in the absence ofantagonists (unpaired t-test with Welch’s correction).

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Figure 34. Effects of 5-HT receptor antagonists on 5-HT-induced contractions of the strips from corpus of the rat stomach. WAY-100635 (A), GR127935 (B), methysergide (C), ketanserin (D), RS-127445 (E), ondansetron (F), SB-203186 (G) or SB-269970 (H) was applied 10 min before addition of 5-HT. The contractions induced by 5-HT was represented as their amplitude relative to amplitude of the contraction induced by 10-5 M carbachol. Each point represents the mean ± S.E.M. from 7-10 animals. *: P<0.05, **: P<0.01, compared with the value in the absence of antagonists (unpaired t-test with Welch’s correction).

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Figure 35. Effect of cinanserin on 5-HT-induced contractions of the stripsfrom corpus of the rat stomach. Cinanserin was applied 10 min beforeaddition of 5-HT. The contractions induced by 5-HT was represented as theiramplitude relative to amplitude of the contraction induced by 10-5 Mcarbachol. Each point represents the mean ± S.E.M. from 8-10 animals. *:P<0.05, compared with the value in the absence of antagonists (unpairedt-test with Welch’s correction).

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4. Discussion

Exogenously applied 5-HT caused contractions of the circular muscle strips from the rat gastric antrum, corpus and fundus. The fundic strips showed higher sensitivity to 5-HT than those of antrum and corpus. Similar results were reported in the circular muscle rings from the mouse stomach (86). The fundus showed higher sensitivity to 5-HT than the antrum. Regional differences of 5-HT receptor distribution might cause the observed regional sensitivity differences. The contractile responses of the antral strips to 5-HT at concentrations of less than 3x10-6 M were enhanced by TTX. Atropine tended to inhibit gastric antral contractions at low concentrations of 5-HT, although this did not reach statistical significance. From these results, in rat gastric antral circular muscle, contractile responses to low concentrations of 5-HT could be mediated through postganglionic cholinergic neurons and regulated by inhibitory neurons. On the other hand, antral contractions to high concentrations of 5-HT were not blocked by atropine nor TTX, indicating the participation of 5-HT receptors directly located on circular smooth muscle. Furthermore, contractile responses to 5-HT in the corporal and fundic strips were also resistant to both atropine and TTX. Based on these results it is likely that corporal and fundic contractions to exogenous 5-HT were mediated via 5-HT receptors located directly on the circular smooth muscle. The regional and functional differences of 5-HT in circular muscle strips from guinea pig stomach have been studied (80, 81). In guinea pigs, 5-HT evoked contractile responses in the antrum and fundus, whilst relaxant responses in the corpus. According to these reports, antral contractions were mediated through neuronal stimulation alone (80) or a combination of both neuronal and smooth muscle stimulations (81). TTX inhibited contractile responses to 5-HT in the guinea pig antrum (80, 81), suggesting the existence of different neuronal mechanisms between rats and guinea pigs. In the rat colon, TTX enhanced circular muscle contractions (87), indicating the attribution of inhibitory neurons such as nitric oxide (NO)-releasing neuron (87, 88) and vasoactive intestinal polypeptide (VIP)-containing neuron (87). In this chapter, a NO synthetase inhibitor, L-NOARG enhanced contractile responses to 5-HT, suggesting NO-releasing inhibitory neurons may be involved and dominant in the rat antrum. On the other hand, the excitatory neurons are dominant in the guinea pig antrum. Furthermore, in guinea pigs, the effects of 5-HT on the corpus and fundus were attributed to stimulation of 5-HT receptors on the smooth muscle. The suggested mechanisms of 5-HT on the corpus and fundus are similar in both rats and guinea pigs, although functions observed in the corpus are different.

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In the antrum, the 5-HT-induced contractions were significantly inhibited by methysergide (5-HT1, 5-HT2, 5-ht5, 5-HT6 and 5-HT7 receptor antagonist), however contractions were not inhibited by either WAY-100635 (selective 5-HT1A receptor antagonist), GR127935 (5-HT1B/1D receptor antagonist) or SB-269970 (selective 5-HT7 receptor antagonist), suggesting the involvement of 5-HT2, 5-ht5 and 5-HT6 receptors. The pKi values of methysergide to rat 5-ht5 and 5-HT6 receptors were reported to be smaller than 7 (89-91), therefore an inhibitory effect at 5-ht5 and 5-HT6 receptors by methysergide at 10-7 M is unlikely. RS-127445 (selective 5-HT2B receptor antagonist) slightly inhibited and 10-6 M ketanserin (non-selective 5-HT2 receptor antagonist) tended to inhibit the antral contractile responses to 5-HT, although these effects were not statistically significant. Furthermore, distribution of 5-HT2C receptors has been limited to the CNS and choroid plexus (71). These results suggested that 5-HT-induced contractions of the gastric antral circular muscles were mediated through 5-HT2A and 5-HT2B receptors. Consistent with this result, the involvement of 5-HT2 receptors in gastric antral muscle contractions induced by 5-HT was reported in guinea pigs (80, 81) and mice (86). In addition, Takemura et al. (81) reported the involvement of 5-HT3 and 5-HT4 receptors on contractile responses and 5-HT1 receptors on relaxant responses in the guinea pig antrum, and James et al. (86) reported the involvement of 5-HT4 receptors on contractile responses in the mouse antrum. Contradictory to these reports, in this chapter, 5-HT-induced contractions were significantly enhanced by SB-203186 (selective 5-HT4 receptor antagonist), suggesting the involvement of 5-HT4 receptors for antral relaxations. This discrepancy was also demonstrated between the ileum strips from guinea pigs (24) and rats (92, 93). In the rat ileum, 5-HT4 receptors are located on the smooth muscles and promote cAMP formation (71), resulting in the relaxation. Our results are consistent with earlier findings that a selective 5-HT4 receptor antagonist, SB 204070 tended to enhance the contractions to 5-HT in the rat ileum (93).

In the corpus, the contractile responses to 5-HT were significantly inhibited by methysergide and ketanserin. However contractions were not inhibited by WAY-100635, GR127935, RS-127445 or SB-269970, suggesting the involvement of 5-HT2A receptors in the contraction. The effect of 5-HT on rat corporal strips remains to be fully investigated. However, platelet-activating factor (PAF)-induced corporal contractions were blocked by methysergide, indicating the involvement of 5-HT in the rat corporal contraction (94). Janssen et al. (95, 96) have shown the mediation of contractions in canine and porcine proximal stomach longitudinal smooth muscles to involve 5-HT2A receptors, which is consistent with the present results. On the other hand, in guinea pig corpus, 5-HT caused relaxations that were mediated via 5-HT1 receptors (79-81). In this

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chapter, pretreatment with WAY-100635 or GR127935 did not demonstrate apparent effect on 5-HT-induced contractions, but tended to enhance the contraction. It is probable that 5-HT1 receptors cause relaxations in the rat corpus, but its functional contribution on these responses to 5-HT is limited. Ondansetron also tended to enhance the 5-HT-induced contractions, implying that 5-HT3 receptors cause relaxations in the rat corpus.

In the fundus, the 5-HT-evoked contractions were significantly inhibited by methysergide and RS-127445, but were not antagonized by WAY-100635, GR127935 or ketanserin, suggesting the involvement of 5-HT2B receptors for the contraction. 5-HT2B receptors were first identified in rat stomach fundus (97, 98), and it is generally accepted that 5-HT2B receptors mediate rat fundic longitudinal smooth muscle contractions (67-69, 99). We have confirmed that 5-HT caused contractions through 5-HT2B receptors not only in longitudinal strips but also in circular ones. Furthermore, 10-7 M ketanserin tended to enhance contractile responses to 5-HT, but 10-6 M ketanserin did not influence the contraction. The pA2 value of ketanserin at 5-HT2B receptors in rat gastric fundic longitudinal muscles is less than 6.0 (99) and the pKB values of ketanserin at human 5-HT2A and 5-HT2B receptors expressed cells are 8.7 and 6.0, respectively (100). It is likely that 5-HT2B receptors antagonism of 10-6 M ketanserin could counteract the enhancement of 5-HT-induced contraction via its 5-HT2A receptors antagonism. In order to confirm the involvement of 5-HT2A receptors in 5-HT-induced fundic relaxations, cinanserin, which is a selective 5-HT2 receptor antagonist showing low affinity to 5-HT2B binding sites (9) was applied. Application of 10-6 M cinanserin significantly enhanced contractile responses to 5-HT, indicating the presence of 5-HT2A receptor-mediated relaxations in this tissue. In addition, SB-203186 also increased 5-HT-caused fundic contractions, suggesting the involvement of 5-HT4 receptors for the relaxations. Amemiya et al. (68) reported that 5-HT4 receptors mediated contractions in the rat fundus. The conflicting results could at least in part be due to the differences in smooth muscle preparations between circular muscles and longitudinal ones. In the earlier findings, 5-methoxytryptamine induced the rat fundic contractions of the longitudinal strips, and these responses were antagonized by atropine, TTX and SDZ 205-557 (5-HT4 receptor antagonist). These results suggested that the longitudinal muscle contractions were mediated through 5-HT4 receptors located on cholinergic neurons. This contractile mechanism is similar to the case of an enhancement of electrically-evoked contractions by 5-HT4 agonists. On the other hand, the present study suggested that the fundic circular muscle relaxations were mediated through 5-HT4 receptors located on smooth muscle. Similar results are reported in the

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rat ileum (92) and the rat esophagus (37, 101). In the tunica muscularis mucosae of the rat esophagus, 5-HT4 receptors stimulated cAMP formation results in the relaxation (101). Similar to 5-HT4 receptors, 5-HT7 receptors coupled to Gs protein activate cAMP formation and are expressed in the rat fundus (102), supporting the fundic relaxation via 5-HT7 receptors. In the present study, SB-269970 tended to enhance the 5-HT-induced fundic contractions. It is probable that a relaxation pathway involving 5-HT7 receptors is present in the rat fundus.

In Chapter II, effects of 5-HT-related compounds had been investigated using in vivo rat gastric emptying and gastric motility models. A 5-HT2 receptor agonist, α-methyl-5-HT stimulated gastric motility. The contractile 5-HT2A and 5-HT2B receptors identified in antral, corporal and fundic circular muscles may play a role in the in vivo contractile activities. The gastrokinetic effect of 5-HT4 agonists also demonstrated in rats. In this chapter, the relaxant 5-HT4 receptors were identified in antral and fundic circular muscles. In the rat stomach, 5-HT4 receptors located on cholinergic neurons may increase acetylcholine release that enhances the contractions similar as in guinea pigs (25). Thus, this contractile mechanism may dominantly occur, though the circular muscle relaxation is also observed. Furthermore, ondansetron accelerated gastric empting but did not change gastric antral motility. Ondansetron tended to enhance contractile responses to 5-HT in the corpus. This enhanced contraction may play a role in part to accelerate gastric emptying.

In conclusion, the main finding of this chapter is that 5-HT mediates contractile responses in rat stomach circular muscle through multiple 5-HT receptor subtypes. 5-HT induces contractions of the circular muscle strips from all three gastric regions. The sensitivity to 5-HT of the fundus is higher than those of antrum and corpus. Contractile responses to 5-HT in the antrum are mediated by 5-HT receptors on smooth muscle and neurons and in the corpus and fundus are mainly mediated by 5-HT receptors located directly on smooth muscle. At least three receptor subtypes mediate the contractile responses observed in rat stomach circular muscles. The contractile 5-HT2A and 5-HT2B receptors and the relaxant 5-HT4 receptors in the antrum, the contractile 5-HT2A receptors in the corpus, and the contractile 5-HT2B receptors and the relaxant 5-HT2A and 5-HT4 receptors in the fundus may be present. The regional difference of expressed 5-HT receptor subtypes and the contractile responses and affinities to 5-HT might cause the regional difference of the sensitivity and the efficacy to 5-HT. This chapter has clarified that contractile responses to 5-HT present regional and functional differences in rat stomach circular muscle, which are species-specific. The regional differences of the sensitivity to 5-HT may be regulated by several 5-HT

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receptor subtypes which modulate gastric emptying in a cooperative way in rats.

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Chapter IV: Effects of 5-HT4 receptor agonists on contractile responses of circular muscle from the rat stomach 1. Introduction

As shown in Chapter III, regional and functional differences to 5-HT in rat gastric circular muscles are characterized. In the antrum, the contractile responses to 5-HT are mediated by 5-HT receptors on both smooth muscle and neurons. 5-HT induces contractions via 5-HT2A and 5-HT2B receptors and relaxations via 5-HT4 receptors. In the corpus, 5-HT induces contractions via 5-HT2A receptors on smooth muscle. In the fundus, the contractile responses to 5-HT are mediated by 5-HT receptors on smooth muscle. 5-HT induces contractions via 5-HT2B receptors and relaxations via 5-HT2A and 5-HT4 receptors.

Other than 5-HT4 agonism, it is reported that cisapride enhanced acetylcholine release from guinea pig ileum due to the blockade of 5-HT1 receptors, but inhibited acetylcholine release from guinea pig ileum due to the blockade of 5-HT3 receptors (25). 5-HT2A antagonism of cisapride was suggested in longitudinal muscles from the rat ileum (93) and canine colon (103). With regard to tegaserod, 5-HT2B receptor antagonistic activity was reported in rat fundic strips (39). Other than these reports, pharmacological characterization of cisapride, mosapride and tegaserod to 5-HT receptor subtypes are limited.

In this chapter, in order to differentiate pharmacological profiles among the 5-HT4 agonists, effects of cisapride, mosapride, tegaserod and CJ-033466 on rat gastric circular muscle of the antrum, corpus and fundus were investigated. 2. Materials and Methods

Methods for preparation of smooth muscle strips from the rat stomach and measurement of mechanical activity are as same as Chapter III. Briefly, Single doses of agonists were applied and then washed out on an interval of 30 to 35 min. To evaluate the effects of 5-HT4 agonists on 5-HT-induced contractions or the effects of SB-203186 on 5-HT4 agonist-induced contractile responses, the tissue strips were exposed to the 5-HT4 agonists or SB-203186 10 min before and during application of 5-HT or the 5-HT4 agonist, respectively. To evaluate the effects of 5-HT4 agonists on α-methyl-5-HT-induced contractions, the tissue strips were exposed to ketanserin (10-7 M) or RS-127445 (10-7 M) 12 min before and 5-HT4 agonists 10 min before and during

- 63 -

application of α-methyl-5-HT. Amplitudes of contraction and relaxation are represented relative to amplitude of contraction elicited by 10-5 M carbachol. Chemicals

CCh was purchased from Wako Pure Chemicals (Osaka, Japan). 5-HT, α-methyl-5-HT maleate and ketanserin tartrate were purchased from Sigma (St. Louis, MO, U.S.A.). CJ-033466, cisapride, mosapride, tegaserod, SB-203186 and RS-127445 were synthesized at Pfizer Inc.

CCh, 5-HT, α-methyl-5-HT and ketanserin were dissolved in distilled water to produce 10-2 or 10-3 M and then diluted to the required concentrations with Krebs-Ringer solution. RS-127445 was dissolved in dimethyl sulfoxide to produce 10-3 M and then diluted to the required concentrations with distilled water. For concentration-response curve study, mosapride and tegaserod were dissolved in 40% sulfobutylether-β-cyclodextrin (SBECD) to produce 10-2 M and diluted to 10-3 M with distilled water and then diluted to the required concentrations with Krebs-Ringer solution. Cisapride and CJ-033466 were dissolved in 1% lactic acid to produce 10-2 M and diluted to 10-3 M with distilled water and then diluted to the required concentrations with Krebs-Ringer solution. For examining the effect on contractile responses to 5-HT and α-methyl-5-HT, mosapride and tegaserod were dissolved in 40% SBECD to produce 10-2 M and diluted to 10-3 M with 4% SBECD. Cisapride and CJ-033466 were dissolved in 1% lactic acid to produce 10-2 M and then diluted to the required concentrations with 1% lactic acid. Statistics

Data were presented as mean ± S.E.M. The comparison between the control group and the antagonist-treated group was carried out by unpaired t-test with Welch's correction. P values of less than 0.05 were regarded as significant. 3. Results Effects of 5-HT4 receptor agonists on the antral, corporal and fundic circular muscle. Cisapride evoked contractions in gastric antral strips at 3x10-6 M to 3x10-5 M. The maximal response was observed at a concentration of 10-5 M. Cisapride did not induce marked responses in the corpus and fundus (Fig. 36). Mosapride relaxed gastric corporal strips at concentrations higher than 10-5 M, but did not induce marked responses in gastric antral and fundic strips (Fig. 37). Tegaserod evoked marked

- 64 -

contractions in gastric antral, corporal and fundic strips at a concentration of 10-4 M (Fig. 38). CJ-033466 evoked contractions in gastric corporal strips at the concentration of 10-4 M. CJ-033466 did not induce marked responses in the antrum, but tended to relax in the fundus (Fig. 39). Effects of SB-203186 on the 5-HT4 receptor agonists-induced contractile responses. Pretreatment of SB-203186 (10-6 M) did not block the cisapride (10-5 M)-evoked contraction in the antrum, mosapride (10-4 M)-induced relaxation in the corpus, tegaserod (10-4 M)-evoked contractions in the fundus or CJ-033466 (10-4 M)-evoked contractions in the corpus (Fig. 40). Effects of 5-HT4 receptor agonists on the 5-HT-induced contractions of the antral, corporal and fundic circular muscle. Pretreatment with cisapride (10-6 M) tended to inhibit contractile responses to 5-HT (3 x 10-6 M), whilst enhanced contractile responses to 5-HT (3 x 10-4 M) of the antral strips. Cisapride inhibited 5-HT-induced contractions of the corporal and fundic strips (Fig. 41). Pretreatment with mosapride (10-6 M) inhibited 5-HT-induced contractions of the fundic strips. Mosapride tended to inhibit 5-HT-induced contractions of the antral strips, but the inhibitory effect was not statistically significant. Mosapride had no effect on the 5-HT-induced contractions of the corporal strips (Fig. 42). Pretreatment of tegaserod (10-6 M) inhibited 5-HT-induced contractions of the fundic strips. Tegaserod tended to inhibit 5-HT-induced contractions of the antral strips, but the inhibitory effect was not statistically significant. Tegaserod had no effect on the 5-HT-induced contractions of the corporal strips (Fig. 43). Pretreatment of CJ-033466 (10-8 and 10-6 M) had no effect on the 5-HT-induced contractions of the antral, corporal and fundic strips (Fig. 44). Effects of 5-HT4 receptor agonists on the α-methyl-5-HT-induced contractions of the antral, corporal and fundic circular muscle. Alpha-methyl-5-HT evoked concentration-dependent contraction of the strips from antrum, corpus and fundus (Figs 45, 46). Cisapride (10-6 M) inhibited α-methyl-5-HT-induced contractions of the corporal strips under the treatment with RS-127445 (Fig.45A). Cisapride (10-6 M), mosapride (10-6 M) or tegaserod (10-6 M) inhibited α-methyl-5-HT-induced contractions of the fundic strips under the treatment with ketanserin (Fig. 45B - D). In the antral strips, cisapride had no effect on α-methyl-5-HT-induced contractions under the treatment with RS-127445, but tended to

- 65 -

inhibit that under the treatment with ketanserin (Fig. 46A, B). Mosapride tended to enhance α-methyl-5-HT-induced contractions under the treatment with ketanserin, but one of 6 strips exhibited strong contractions. Except for it, concentration-response curves for α-methyl-5-HT in the absence of mosapride was comparable with that in the presence of mosapride (Fig. 46C). Tegaserod had no effect on α-methyl-5-HT-induced contractions under the treatment with ketanserin (Fig. 46D).

- 66 -

% c

ontr

actio

n of

CC

h1x

10-5

M

(A) Antrum

-8 -7 -6 -5 -40

5

10

15

Log M [Cisapride]

% c

ontr

actio

n of

CC

h1x

10-5

M

(B) Corpus

-8 -7 -6 -5 -4-5

0

5

10

15

Log M [Cisapride]

% c

ontr

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CC

h1x

10-5

M

(C) Fundus

-8 -7 -6 -5 -4-5

0

5

10

Log M [Cisapride]

% c

ontr

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n of

CC

h1x

10-5

M

(A) Antrum

-8 -7 -6 -5 -40

5

10

15

Log M [Cisapride]

% c

ontr

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n of

CC

h1x

10-5

M

(A) Antrum

-8 -7 -6 -5 -40

5

10

15

Log M [Cisapride]-8 -7 -6 -5 -4

0

5

10

15

Log M [Cisapride]

% c

ontr

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CC

h1x

10-5

M

(B) Corpus

-8 -7 -6 -5 -4-5

0

5

10

15

Log M [Cisapride]

% c

ontr

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n of

CC

h1x

10-5

M

(B) Corpus

-8 -7 -6 -5 -4-5

0

5

10

15


-5

0

5

10

15

Log M [Cisapride]

% c

ontr

actio

n of

CC

h1x

10-5

M

(C) Fundus

-8 -7 -6 -5 -4-5

0

5

10

Log M [Cisapride]

% c

ontr

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n of

CC

h1x

10-5

M

(C) Fundus

-8 -7 -6 -5 -4-5

0

5

10


-5

0

5

10

Log M [Cisapride]

Figure 36. Concentration-response curves for cisapride of the strips from antrum(A), corpus (B), and fundus (C) of the rat stomach. The contractions induced by cisapride was represented as their amplitude relative to amplitude of the contractioninduced by 10-5 M carbachol. Each point represents the mean ± S.E.M. from 5 animals.

- 67 -

% c

ontr

actio

n of

CC

h1x

10-5

M

(A) Antrum

-8 -7 -6 -5 -40

5

10

15

Log M [Mosapride]

% c

ontr

actio

n of

CC

h1x

10-5

M

(B) Corpus

(C) Fundus

-8 -7 -6 -5 -4-10

-5

0

5

10

Log M [Mosapride]

% c

ontr

actio

n of

CC

h1x

10-5

M

-8 -7 -6 -5 -4-5

0

5

10

Log M [Mosapride]

% c

ontr

actio

n of

CC

h1x

10-5

M

(A) Antrum

-8 -7 -6 -5 -40

5

10

15

Log M [Mosapride]

% c

ontr

actio

n of

CC

h1x

10-5

M

(A) Antrum

-8 -7 -6 -5 -40

5

10

15

Log M [Mosapride]-8 -7 -6 -5 -4

0

5

10

15

Log M [Mosapride]

% c

ontr

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n of

CC

h1x

10-5

M

(B) Corpus

(C) Fundus

-8 -7 -6 -5 -4-10

-5

0

5

10

Log M [Mosapride]

% c

ontr

actio

n of

CC

h1x

10-5

M

(B) Corpus

(C) Fundus

-8 -7 -6 -5 -4-10

-5

0

5

10


-10

-5

0

5

10

Log M [Mosapride]

% c

ontr

actio

n of

CC

h1x

10-5

M

-8 -7 -6 -5 -4-5

0

5

10

Log M [Mosapride]

% c

ontr

actio

n of

CC

h1x

10-5

M

-8 -7 -6 -5 -4-5

0

5

10


-5

0

5

10

Log M [Mosapride]

Figure 37. Concentration-response curves for mosapride of the strips from antrum(A), corpus (B), and fundus (C) of the rat stomach. The contractions induced by mosapride was represented as their amplitude relative to amplitude of thecontraction induced by 10-5 M carbachol. Each point represents the mean ± S.E.M. from 5 animals.

- 68 -

% c

ontr

actio

n of

CC

h1x

10-5

M

(A) Antrum

-8 -7 -6 -5 -40

5

10

15

Log M [Tegaserod]

% c

ontr

actio

n of

CC

h1x

10-5

M

(B) Corpus

-8 -7 -6 -5 -40

5

10

15

20

Log M [Tegaserod]

% c

ontr

actio

n of

CC

h1x

10-5

M

(C) Fundus

-8 -7 -6 -5 -40

5

10

15

Log M [Tegaserod]

% c

ontr

actio

n of

CC

h1x

10-5

M

(A) Antrum

-8 -7 -6 -5 -40

5

10

15

Log M [Tegaserod]

% c

ontr

actio

n of

CC

h1x

10-5

M

(A) Antrum

-8 -7 -6 -5 -40

5

10

15

Log M [Tegaserod]-8 -7 -6 -5 -4

0

5

10

15

Log M [Tegaserod]

% c

ontr

actio

n of

CC

h1x

10-5

M

(B) Corpus

-8 -7 -6 -5 -40

5

10

15

20

Log M [Tegaserod]

% c

ontr

actio

n of

CC

h1x

10-5

M

(B) Corpus

-8 -7 -6 -5 -40

5

10

15

20


0

5

10

15

20

Log M [Tegaserod]

% c

ontr

actio

n of

CC

h1x

10-5

M

(C) Fundus

-8 -7 -6 -5 -40

5

10

15

Log M [Tegaserod]

% c

ontr

actio

n of

CC

h1x

10-5

M

(C) Fundus

-8 -7 -6 -5 -40

5

10

15


0

5

10

15

Log M [Tegaserod]

Figure 38. Concentration-response curves for tegaserod of the strips from antrum (A), corpus (B), and fundus (C) of the rat stomach. The contractions induced by tegaserod was represented as their amplitude relative to amplitude of the contraction induced by 10-5 M carbachol. Each point represents the mean ± S.E.M. from 5 animals.

- 69 -

% c

ontr

actio

n of

CC

h1x

10-5

M

(A) Antrum

-8 -7 -6 -5 -4-5

0

5

10

Log M [CJ-033466]

% c

ontr

actio

n of

CC

h1x

10-5

M

(B) Corpus

-8 -7 -6 -5 -4-5

0

5

10

15

Log M [CJ-033466]

% c

ontr

actio

n of

CC

h1x

10-5

M

(C) Fundus

-8 -7 -6 -5 -4-5

0

5

10

Log M [CJ-033466]

% c

ontr

actio

n of

CC

h1x

10-5

M

(A) Antrum

-8 -7 -6 -5 -4-5

0

5

10

Log M [CJ-033466]

% c

ontr

actio

n of

CC

h1x

10-5

M

(A) Antrum

-8 -7 -6 -5 -4-5

0

5

10

Log M [CJ-033466]-8 -7 -6 -5 -4

-5

0

5

10

-8 -7 -6 -5 -4-5

0

5

10

Log M [CJ-033466]

% c

ontr

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n of

CC

h1x

10-5

M

(B) Corpus

-8 -7 -6 -5 -4-5

0

5

10

15

Log M [CJ-033466]

% c

ontr

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n of

CC

h1x

10-5

M

(B) Corpus

-8 -7 -6 -5 -4-5

0

5

10

15

Log M [CJ-033466]-8 -7 -6 -5 -4

-5

0

5

10

15

Log M [CJ-033466]

% c

ontr

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n of

CC

h1x

10-5

M

(C) Fundus

-8 -7 -6 -5 -4-5

0

5

10

Log M [CJ-033466]

% c

ontr

actio

n of

CC

h1x

10-5

M

(C) Fundus

-8 -7 -6 -5 -4-5

0

5

10

Log M [CJ-033466]-8 -7 -6 -5 -4

-5

0

5

10

Log M [CJ-033466]

Figure 39. Concentration-response curves for CJ-033466 of the strips from antrum (A), corpus (B), and fundus (C) of the rat stomach. The contractions induced by CJ-033466 was represented as their amplitude relative to amplitude of thecontraction induced by 10-5 M carbachol. Each point represents the mean ± S.E.M. from 5 animals.

- 70 -

(A) Antrum

Cis 10-5 M 10-5 MSB 10-6 M

0

10

20

30

40

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60

% c

ontr

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CC

h1x

10-5

M%

con

trac

tion

of C

Ch

1x10

-5M

Teg 10-4 M 10-4 MSB 10-6 M

(C) Fundus

0

10

20

30

40

50

60

70

% c

ontr

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CC

h1x

10-5

M

Mos 10-4 M 10-4 MSB 10-6 M

(B) Corpus

-20

-15

-10

-5

0

% c

ontr

actio

n of

CC

h1x

10-5

M

CJ 10-4 M 10-4 MSB 10-6 M

(D) Corpus

0

5

10

15

20

(A) Antrum

Cis 10-5 M 10-5 MSB 10-6 M

0

10

20

30

40

50

60

% c

ontr

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n of

CC

h1x

10-5

M(A) Antrum

Cis 10-5 M 10-5 MSB 10-6 M

0

10

20

30

40

50

60

0

10

20

30

40

50

60

10

20

30

40

50

60

% c

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CC

h1x

10-5

M%

con

trac

tion

of C

Ch

1x10

-5M

Teg 10-4 M 10-4 MSB 10-6 M

(C) Fundus

0

10

20

30

40

50

60

70

% c

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h1x

10-5

M

Teg 10-4 M 10-4 MSB 10-6 M

(C) Fundus

0

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60

70

0

10

20

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60

70

% c

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h1x

10-5

M

Mos 10-4 M 10-4 MSB 10-6 M

(B) Corpus

-20

-15

-10

-5

0

% c

ontr

actio

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CC

h1x

10-5

M

Mos 10-4 M 10-4 MSB 10-6 M

(B) Corpus

-20

-15

-10

-5

0

-20

-15

-10

-5

0

% c

ontr

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CC

h1x

10-5

M

CJ 10-4 M 10-4 MSB 10-6 M

(D) Corpus

0

5

10

15

20

% c

ontr

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CC

h1x

10-5

M

CJ 10-4 M 10-4 MSB 10-6 M

(D) Corpus

0

5

10

15

20(D) Corpus

0

5

10

15

20

0

5

10

15

20

Figure 40. Effects of SB-203186 on cisapride (10-5 M)-induced contractions in the antrum (A), mosapride (10-4 M)-induced relaxations in the corpus (B), tegaserod (10-4 M)-induced contractions in the fundus and CJ-033466 (10-4 M)-induced contractions in the corpus. SB-203186 (10-6 M) was applied 10 min before addition of 5-HT4 agonists. The contractile responses induced by 5-HT4 agonists were represented as their amplitude relative to amplitude of the contraction induced by10-5 M carbachol. Each point represents the mean ± S.E.M. from 3 to 5 animals.

- 71 -

ControlCisapride 10-6 M

-7 -6 -5 -4 -30

10

20

30

40

50

60

Log M [5-HT]

% c

ontr

actio

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CC

h1x

10-5

M

(A) Antrum

**%

con

trac

tion

of C

Ch

1x10

-5M

-7 -6 -5 -4 -30

5

10

15

20

25

Log M [5-HT]

******

(B) Corpus

**

****

*

-9 -8 -7 -6 -50

10

20

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40

50

60

70

Log M [5-HT]

% c

ontr

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n of

CC

h1x

10-5

M

(C) Fundus


-7 -6 -5 -4 -30

10

20

30

40

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60

Log M [5-HT]

% c

ontr

actio

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CC

h1x

10-5

M

(A) Antrum

**

ControlCisapride 10-6 M ControlControlCisapride 10-6 M Cisapride 10-6 M

-7 -6 -5 -4 -30

10

20

30

40

50

60

Log M [5-HT]-7 -6 -5 -4 -3

0

10

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Log M [5-HT]

% c

ontr

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h1x

10-5

M

(A) Antrum

**%

con

trac

tion

of C

Ch

1x10

-5M

-7 -6 -5 -4 -30

5

10

15

20

25

Log M [5-HT]

******

(B) Corpus

% c

ontr

actio

n of

CC

h1x

10-5

M

-7 -6 -5 -4 -30

5

10

15

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Log M [5-HT]

******

-7 -6 -5 -4 -30

5

10

15

20

25

Log M [5-HT]-7 -6 -5 -4 -3

0

5

10

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25

Log M [5-HT]

******

(B) Corpus

**

****

*

-9 -8 -7 -6 -50

10

20

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60

70

Log M [5-HT]

% c

ontr

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h1x

10-5

M

(C) Fundus

**

****

*

-9 -8 -7 -6 -50

10

20

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70

Log M [5-HT]

% c

ontr

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CC

h1x

10-5

M

**

****

*

-9 -8 -7 -6 -50

10

20

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40

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70

Log M [5-HT]

% c

ontr

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CC

h1x

10-5

M

-9 -8 -7 -6 -50

10

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Log M [5-HT]-9 -8 -7 -6 -5

0

10

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50

60

70

Log M [5-HT]

% c

ontr

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n of

CC

h1x

10-5

M

(C) Fundus

Figure 41. Effects of cisapride on 5-HT-induced contractions of the strips from antrum (A), corpus (B), and fundus (C) of the rat stomach. Cisapride was applied 10 min before addition of 5-HT. The contractions induced by 5-HT was represented as their amplitude relative to amplitude of the contraction induced by 10-5 M carbachol. Each point represents the mean ± S.E.M. from 8 animals. *: P<0.05, **: P<0.01, compared with the value in the absence of cisapride (unpaired t-test with Welch’s correction).

- 72 -

ControlMosapride 10-6 M

% c

ontr

actio

n of

CC

h1x

10-5

M

(A) Antrum

-7 -6 -5 -4 -30

10

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Log M [5-HT]

% c

ontr

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n of

CC

h1x

10-5

M

(B) Corpus

-7 -6 -5 -4 -30

5

10

15

20

25

Log M [5-HT]

****

*

% c

ontr

actio

n of

CC

h1x

10-5

M

(C) Fundus

-9 -8 -7 -6 -50

10

20

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Log M [5-HT]

ControlMosapride 10-6 M ControlControlMosapride 10-6 M Mosapride 10-6 M

% c

ontr

actio

n of

CC

h1x

10-5

M

(A) Antrum

-7 -6 -5 -4 -30

10

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Log M [5-HT]

% c

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CC

h1x

10-5

M

(A) Antrum

-7 -6 -5 -4 -30

10

20

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60

Log M [5-HT]-7 -6 -5 -4 -3

0

10

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Log M [5-HT]

% c

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CC

h1x

10-5

M

(B) Corpus

-7 -6 -5 -4 -30

5

10

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Log M [5-HT]

% c

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CC

h1x

10-5

M

(B) Corpus

-7 -6 -5 -4 -30

5

10

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Figure 42. Effects of mosapride on 5-HT-induced contractions of the strips from antrum (A), corpus (B), and fundus (C) of the rat stomach. Mosapride was applied 10 min before addition of 5-HT. The contractions induced by 5-HT was represented as their amplitude relative to amplitude of the contraction induced by10-5 M carbachol. Each point represents the mean ± S.E.M. from 8 animals. *:P<0.05, **: P<0.01, compared with the value in the absence of mosapride (unpaired t-test with Welch’s correction).

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Figure 43. Effects of tegaserod on 5-HT-induced contractions of the strips from antrum (A), corpus (B), and fundus (C) of the rat stomach. Tegaserod was applied 10 min before addition of 5-HT. The contractions induced by 5-HT was represented as their amplitude relative to amplitude of the contraction induced by 10-5 M carbachol. Each point represents the mean ± S.E.M. from 8 animals. *: P<0.05, **: P<0.01, compared with the value in the absence of tegaserod (unpaired t-test with Welch’s correction).

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Figure 44. Effects of CJ-033466 on 5-HT-induced contractions of the strips from antrum (A), corpus (B), and fundus (C) of the rat stomach. CJ-033466 was applied 10 min before addition of 5-HT. The contractions induced by 5-HT was represented as their amplitude relative to amplitude of the contraction induced by10-5 M carbachol. Each point represents the mean ± S.E.M. from 8 animals.

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(A) Corpus


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Figure 45. Effects of 5-HT4 agonists on α-methyl-5-HT-induced contractions of thestrips from corpus (A) and fundus (B - D) of the rat stomach. Cisapride (A, B), mosapride (C) or tegaserod (D) was applied 10 min before addition of α-methyl-5-HT. RS-127445 (10-7 M) for the corpus or ketanserin (10-7 M) for the fundus was applied 12 min before addition of α-methyl-5-HT. The contractionsinduced by α-methyl-5-HT was represented as their amplitude relative to amplitudeof the contraction induced by 10-5 M carbachol. Each point represents the mean ± S.E.M. from 6 animals. *: P<0.05, **: P<0.01, compared with the value in the absence of 5-HT4 agonists (unpaired t-test with Welch’s correction).

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(A) RS-127445 pretreatment


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Figure 46. Effects of 5-HT4 agonists on α-methyl-5-HT-induced contractions of thestrips from antrum of the rat stomach. Cisapride (A, B), mosapride (C) or tegaserod (D) was applied 10 min before addition of α-methyl-5-HT. RS-127445 (10-7 M, A) or ketanserin (10-7 M, B - D) was applied 12 min before addition of α-methyl-5-HT. The contractions induced by α-methyl-5-HT was represented as their amplituderelative to amplitude of the contraction induced by 10-5 M carbachol. Each point represents the mean ± S.E.M. from 6 animals.

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4. Discussion

Pretreatment of SB-203186 (10-6 M) did not block the cisapride (10-5 M)-evoked contractions in the antrum, mosapride (10-4 M)-induced relaxations in the corpus, tegaserod (10-4 M)-evoked contractions in the fundus, nor CJ-033466 (10-4 M)-evoked contractions in the corpus, suggesting these observed effects were induced via other than 5-HT4 receptors. Cisapride is known to stimulate spontaneous motility of isolated ileum and gastroduodenum preparations from the guinea pig (25, 104). Taniyama et al. (25) suggested that at high concentrations of cisapride (10-6 M to 10-5 M)-evoked ileal contractions and the release of acetylcholine were induced via putative 5-HT4 receptors. In the present study, SB-203186 did not block cisapride-evoked spontaneous contraction in the rat antrum. The conflicting results could at least in part be due to the differences in tissue preparations between the guinea pig ileum and the rat antrum. Furthermore, cisapride is a fairly potent HERG K+ channel blocker (105, 106). It is known that HERG K+ channel are expressed on intestinal cells of Cajal which regulates gastrointestinal motor activity (107, 108). The blockade of HERG K+ channel by cisapride may stimulate contractile activities in the antrum. This mechanism also may play a role in the enhancement of 3 x 10-4 M 5-HT-induced contractions by cisapride. Tegaserod (10-4 M) evoked contractions in all the regions, indicating that non-specific mechanisms such as Ca2+ influx may be involved. The relaxant responses to mosapride and contractile responses to CJ-033466 were regional-specific, but pathways that induce these responses still remain unclear.

In the fundus, cisapride, mosapride and tegaserod inhibited contractile responses to 5-HT. 5-HT2B antagonism blocked 5-HT-induced fundic contractions (Fig. 34). Cisapride, mosapride and tegaserod also inhibited contractile responses to α-methyl-5-HT, a 5-HT2 agonist under the treatment with 10-7 M ketanserin which selectively blocked 5-HT2A receptor. These results suggested that not only tegaserod but also cisapride and mosapride may have 5-HT2B receptor antagonistic activities. In rat gastric emptying study, it is suggested that 5-HT2B antagonism of tegaserod may contribute the lack of efficacy on gastric emptying. A high potency of tegaserod to the 5-HT2B receptor may affect on the gastric emptying.

In the corpus, cisapride inhibited 5-HT-induced contractions as similar as those of 5-HT2A receptor antagonists as shown in Fig. 33. Cisapride also inhibited contractile responses to α-methyl-5-HT under the treatment with 10-7 M RS-127445 which selectively blocked 5-HT2B receptor, indicating that cisapride has 5-HT2A antagonism.

In the antrum, cisapride tended to inhibit 3 x 10-6 M 5-HT-induced contractions.

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Mosapride and tegaserod also tended to inhibit 5-HT-induced contractions. These inhibitory effects were not significant statistically, but similar tendency was observed by pretreatment with RS-127445 against 5-HT-induced contractions (Fig. 32), suggesting the involvement of 5-HT2B antagonism of these compounds. On the other hand, contractile responses to α-methyl-5-HT were not inhibited by mosapride and tegaserod under the treatment with ketanserin. From these results, the involvement with 5-HT2B receptor to 5-HT-induced contractions in the antrum may be limited. Cisapride had no effect on α-methyl-5-HT under the treatment with RS-127445, but tended to inhibit α-methyl-5-HT under the treatment with ketanserin. The results from the fundus and corpus, cisapride has both 5-HT2A and 5-HT2B receptor antagonistic activities. Furthermore, a high concentration of ketanserin (10-6 M) demonstrated the tendency to inhibit 5-HT-induced contractions (Fig. 32). These results suggested that high concentration of 5-HT2A receptor antagonists is needed to inhibit contractile responses to 5-HT in the antrum. Thus, 5-HT-induced contractions in the antrum may be dominantly regulated by 5-HT2A receptors than 5-HT2B receptors.

CJ-033466 had no effects on 5-HT-induced contractions in the antrum, corpus and fundus. Differently from cisapride, mosapride and tegaserod, CJ-033466 seems not to have 5-HT2A and 5-HT2B receptor antagonist activities. It is suggested that CJ-033466 may have higher selectivity to 5-HT4 receptors.

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Chapter V: Effects of CJ-033466 on ACh release from human gastric antral tissues 1. Introduction Stimulation of 5-HT4 receptors accelerates the motility of the gastrointestinal tract in vivo, while this stimulation shows excitatory or inhibitory effects on isolated preparations, depending on the species and anatomical region investigated (109, 110). In the isolated human colonic preparations, simulation of 5-HT4 receptors causes relaxations of circular muscles (111). In isolated preparations from rats, simulation of 5-HT4 receptors causes relaxations of esophagus and ileum (37, 111), as is the case in human colonic tissue. On the other hand, in the isolated preparations of stomach (82, 112), ileum (24, 25, 113) and colon (114) from guinea pigs, simulation of 5-HT4 receptors causes contractions due to stimulation of excitatory neurons, such as cholinergic neurons and tachykinin-containing neurons.

In vitro receptor autoradiography of 5-HT4 receptors in the human gastric antrum has demonstrated the localization of 5-HT4 receptors at the myenteric plexus of human gastric antrum (115). In this chapter, the function of 5-HT4 receptor in the human gastric antrum, in relation to the contractility and acetylcholine (ACh) release were examined.

Furthermore, gastrokinetic effects of CJ-033466 have been evaluated as a 5-HT4 agonist comparison with cisapride, mosapride and tegaserod in animal models. In vitro, other than 5-HT4 agonism, CJ-033466 demonstrates the different pharmacological profile from cisapride, mosapride and tegaserod. Therefore, in order to determine whether the action of CJ-033466 can be applied to human as a 5-HT4 agonist, effect of CJ-033466 on ACh release from human gastric antral tissues was examined. 2. Materials and Methods Measurement of the mechanical activity

Specimens of human gastric antrum were obtained from patients undergoing surgical resection for gastric cancer. The normal tissues adjacent to the pathological ones were cut and used. The mucosa was rapidly removed from the tissue. The strips were placed in a 20-mL organ bath in the presence of Krebs-Ringer solution of the following composition: 118 mM NaCl, 4.8 mM KCl, 2.5 mM CaCl2, 1.19 mM MgSO4, 25.0 mM NaHCO3, 1.18 mM KH2PO4 and 11 mM glucose, oxygenated with 95% O2 and 5% CO2 and maintained at 34-36℃. Approximately 1 g of resting tension were applied and were kept constant by re-adjustment during the equilibration period, and then the bath

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medium was changed to Krebs-Ringer solution containing methysergide (10-7 M), ketanserin (10-7 M) and granisetron (10-7 M). Mechanical responses were recorded by means of an isometric transducer (SD-IT, Nihon Koden, Tokyo, Japan). The electrical field stimulation (EFS) was achieved using monophasic square-wave pulses of 4 msec pulse width and 5 Hz frequency at 70 V. EFS was applied for 20 s at 2 min intervals 10 times for 30 min periods. After a stabilization of EFS-induced contraction, 5-HT was applied to the organ in the absence and presence of antagonists for 5-HT4 receptors. Atropine was applied to the same strips at the end of measurement of 5-HT effect. Measurement of [3H]ACh outflow

Three preparations from the gastric antrum were incubated at 35℃ for 60 min with [3H]choline at a final concentration of 200 nM in Krebs solution. After washing in fresh Krebs solution for 15 min, the preparations were mounted in the superfusion apparatus and superfused at 1 mL/min with Krebs-Ringer solution gassed with 95% O2 and 5% CO2, maintained at 35-37℃. Experiments were begun 30 min after superfusion, and the superfusate was collected every 2 min 7 times (fractions) in a series. Two parallel platinum electrodes were used to stimulate intramural nerves. Electrical stimulation (ES) was applied to the preparation at the fourth fraction in a series, and successively 4 times (S1, S2, S3 and S4) to the preparation at 30 min intervals. The conditions of ES were 1 msec pulse width, 3 Hz frequency at 60 V for 2 min. The superfusate was collected every 2 min and the radioactivity of the sample was determined by counting in a liquid scintillation spectrometer. The effect of ES was evaluated by the amounts of [3H]ACh outflow in the fourth fraction against the mean of those in 3 fractions before ES, the first to third fraction. Perfusion medium contained 10-7 M methysergide, 10-7 M ketanserin and 10-7 M granisetron to prevent the effect of 5-HT on the 5-HT1, 5-HT2 and 5-HT3 receptors.

The concentration-dependency of 5-HT was evaluated by comparing the ratio of S3/S2 and S4/S2 in three preparations isolated from the same specimen. In a preparation, 5-HT was absent during S1, S2, S3 and S4 ES. In the other preparations, 5-HT was absent during S1 and S2 ES and present 2 min before and during the S3 and S4 ES.

The effect of 5-HT4 receptor antagonist was also evaluated by comparing the ratio of S3/S2 and S4/S2 in three preparations isolated from the same specimen. In a preparation, 5-HT was absent during S1, S2, S3 and S4 ES. In a preparation, 5-HT at 10-6 M was absent during S1 and S2 ES and present 2 min before and during the S3 and S4 ES. In another preparation, 5-HT at 10-6 M was absent during S1 and S2 ES and present 2 min before and during the S3 and S4 ES, and SB-203186 (5-HT4 receptor antagonist) at 10-6

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M was present during S4 ES. CJ-033466 was applied the similar protocol as the 5-HT experiment, except for

CJ-033466 was absent during S4 for the concentration-dependency experiment. CJ-033466 at 10-7 M was used for the antagonist experiment. Chemicals

[3H]choline (3.33 TBq/mmol) was purchased from New England Nuclear (Boston, MA, USA). 5-HT creatinine sulfate was purchased from Sigma (St. Louis, MO, USA). Methysergide maleate and ketanserin tartrate were purchased from Research Biochemicals Int. (Natick, MA, USA). Atropine sulfate monohydrate and tetrodotoxin were purchased from Wako (Osaka, Japan). SB-203186 and granisetron were purchased from Tocris Cookson Inc (Ellisville, MO, USA). CJ-033466 was synthesized at Pfizer Inc. Statistics

Data were presented as mean ± S.E.M. Data were analyzed by unpaired t-test. P values of less than 0.05 were regarded as significant. 3. Results Effect of 5-HT on electrically stimulated contractions of human gastric antral preparations. EFS (5 Hz, 4 msec) evoked a monophasic contraction over the spontaneous contractions in the antral preparations from the human stomach (Fig. 47A). Application of 10-6 M 5-HT enhanced EFS-induced gastric antral contractions (Fig. 47A). SB-203186 (10-6 M) antagonized the enhanced contractions induced by 5-HT (Fig. 47B). The EFS-induced contractions and 5-HT-induced enhancement of contractions were prevented by 10-6 M atropine (data not shown). Effect of 5-HT on electrically stimulated outflow of [3H]ACh from human gastric antral preparations. ES (3 Hz, 60 V, 1 msec, for 2 min) evoked the outflow of [3H]ACh over the spontaneous [3H]ACh outflow from the mucosa-free preparations of the human gastric antrum. The ES-evoked outflow of [3H]ACh was prevented by 3 x 10-7 M TTX (data not shown), indicating that the released [3H] originates from the nerve terminals. ES was applied 4 times every 30 min to the same preparation. The ratio of S3-evoked

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[3H]ACh outflow / S2-evoked [3H]ACh outflow and S4-evoked [3H]ACh outflow / S2-evoked [3H]ACh outflow were respectively compared in three preparations from the same donor. There were no significant differences in the ratio of S3-evoked [3H]ACh outflow / S2-evoked [3H]ACh outflow and S4-evoked [3H]ACh outflow / S2-evoked [3H]ACh outflow in the absence of 5-HT.

5-HT at 10-8 M to 10-5 M potentiated the ES-evoked outflow of [3H]ACh from the preparations of human antrum, in a concentration-dependent manner (Fig. 48A). SB-203186 (10-6 M) completely antagonized 5-HT (10-6 M)-induced potentiation (Fig. 48B). Effect of CJ-033466 on electrically stimulated outflow of [3H]ACh from human gastric antral preparations.

CJ-033466 at 10-9 M to 10-7 M potentiated the ES-evoked outflow of [3H]ACh from the preparations of human antrum, in a concentration-dependent manner (Fig. 49A). The maximal potentiation by CJ-033466 was reached at a concentration of 10-7 M. CJ-033466 at 10-6 M induced weaker potentiation than that observed at 10-7 M. SB-203186 (10-6 M) completely antagonized CJ-033466 (10-7 M)-induced potentiation (Fig. 49B).

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5-HT 10-6 M

(A)

5-HT 10-6 MSB-203186 10-6 M

(B)

5-HT 10-6 M

(A)

5-HT 10-6 M5-HT 10-6 M

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5-HT 10-6 MSB-203186 10-6 M

(B)

5-HT 10-6 MSB-203186 10-6 M 5-HT 10-6 MSB-203186 10-6 M 5-HT 10-6 MSB-203186 10-6 M

(B)

Figure 47. Typical pattern of enhancement of electrical field stimulation(EFS)-induced contraction by 5-HT at 10-6 M (A) and inhibitory effect of SB-203186 (10-6 M) on 5-HT-enhanced contraction (B) in the presence of methysergide (10-7 M), ketanserin (10-7 M) and granisetron (10-7 M).

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1.0

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― + + 5-HT 10-6 M― ― + SB 10-6 M

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Figure 48. Concentration-response curves for 5-HT4 receptor-mediated potentiationof electrically stimulated [3H]ACh outflow (A, n=2) and effect of SB-203186 on the 5-HT4 receptor-mediated potentiation of electrically stimulated [3H]ACh outflow (B, n=3) from the gastric antrum isolated from human. Electrical stimulation (3 Hz, 1 msec, 60 V) was applied for 2 min in the presence of methysergide (10-7 M), ketanserin (10-7 M) and granisetron (10-7 M). The [3H]ACh outflow was represented as a ratio of the release evoked by the third or fourth stimulation (S3 or S4) to thatby the second stimulation (S2). 5-HT was present during the third and fourth stimulation (S3, S4). SB-203186 was present during the fourth stimulation (S4). Each point represents the mean ± S.E.M. #: P<0.05, compared with control value. *: P<0.05, compared with the value in the absence of SB-203186 (Unpairedt-test).

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― + SB 10-6 MCJ 10-7 M

*

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Figure 49. Concentration-response curves for CJ-033466-mediated potentiation ofelectrically stimulated [3H]ACh outflow (A, n=2-3) and effect of SB-203186 on the CJ-033466-mediated potentiation of electrically stimulated [3H]ACh outflow (B,n=3) from the gastric antrum isolated from human. Electrical stimulation (3 Hz, 1 msec, 60 V) was applied for 2 min in the presence of methysergide (10-7 M), ketanserin (10-7 M) and granisetron (10-7 M). The [3H]ACh outflow was represented as a ratio of the release evoked by the third or fourth stimulation (S3 or S4) to thatby the second stimulation (S2). CJ-033466 was present during the third and/or fourthstimulation (S3, S4). SB-203186 was present during the fourth stimulation (S4). Each point represents the mean ± S.E.M. *: P<0.05, compared with the value inthe absence of SB-203186 (Unpaired t-test).

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4. Discussion 5-HT enhanced EFS-evoked contractions which were antagonized by atropine. The responses to 5-HT were obtained in the preparations under the 5-HT1, 5-HT2 and 5-HT3 receptors blockade. This enhancement was prevented by the 5-HT4 receptor antagonist. These results indicate that stimulation of 5-HT4 receptors accelerates the cholinergic transmission in the human gastric antrum. Application of ES to the preparations preloaded with [3H]choline evoked the [3H] outflow. This [3H] outflow was prevented by TTX. Thus this [3H] outflow may probably be the [3H]ACh originates from nerve terminals. 5-HT enhanced ES-evoked [3H]ACh outflow which was antagonized by SB-203186 under the application of 5-HT1, 5-HT2 and 5-HT3 receptor antagonists. Therefore, 5-HT enhanced ES-evoked [3H]ACh outflow via stimulation of 5-HT4 receptors. The results obtained in the human gastric antrum were similar to those in the guinea pig antrum (116). There is a possibility that the 5-HT4 receptors noted on the myenteric plexus in the human gastric antrum by receptor autoradiography (115) are located on the cholinergic neurons. Therefore, stimulation of 5-HT4 receptors located on the myenteric plexus of human gastric antrum accelerates the ACh release from the cholinergic nerve terminals, and may stimulate the motor activity of gastric antrum. The responses to 5-HT observed in the human gastric antrum are different from the findings in the isolated human colonic preparations, in which stimulation of 5-HT4 receptors induced relaxations of circular muscles (111). This difference could at least in part be due to the location dominantly expressed 5-HT4 receptors in tissue, on neurons or on smooth muscles. CJ-033466 enhanced ES-evoked [3H]ACh outflow which was antagonized by SB-203186 under the application of 5-HT1, 5-HT2 and 5-HT3 receptor antagonists. These results indicate that CJ-033466 enhanced ES-evoked [3H]ACh outflow via stimulation of 5-HT4 receptors. Therefore, CJ-033466 stimulates 5-HT4 receptors located on the myenteric plexus, and accelerates the ACh release from the cholinergic nerve terminals, and may express gastrokinetic effects in the human stomach.

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Concluding Remarks In the present study, CJ-033466 demonstrated gastrokinetic actions in all animal models, stimulating canine gastric motility both under fasted and postprandial states, accelerating rat gastric emptying and stimulating rat gastric motility via stimulation of 5-HT4 receptors. Other than CJ-033466, only cisapride demonstrated gastrokinetic actions in all animal models. CJ-033466 is 30 times more potent on canine gastric motility and it is 3 times less potent on rat gastric emptying than those of cisapride. CJ-033466 may not have 5-HT2A and 5-HT2B receptor antagonistic activities which cisapride, mosapride and tegaserod have. CJ-033466 also stimulated 5-HT4 receptors located on the myenteric plexus of human gastric antrum which may accelerate the ACh release from the cholinergic nerve terminals, and may stimulate the motor activity of gastric antrum. In rat stomach circular muscle, regional and functional differences of 5-HT receptor subtypes has been identified. In the antrum, the contractile responses to 5-HT are mediated by 5-HT receptors on both smooth muscle and neurons. 5-HT induces contractions via 5-HT2A and 5-HT2B receptors and relaxations via 5-HT4 receptors. In the corpus, 5-HT induces contractions via 5-HT2A receptors on smooth muscle. In the fundus, the contractile responses to 5-HT are mediated by 5-HT receptors on smooth muscle. 5-HT induces contractions via 5-HT2B receptors and relaxations via 5-HT2A and 5-HT4 receptors. Contractile responses to 5-HT in the rat stomach circular muscle present regional and functional differences, which are species-specific. 5-HT induces contractions of the circular muscle strips from all three gastric regions. The sensitivity to 5-HT of the fundus is higher than those of antrum and corpus. The regional differences of the sensitivity to 5-HT may be regulated by several 5-HT receptor subtypes which modulate gastric emptying in a cooperative way in rats. CJ-033466 and cisapride demonstrated gastrokinetic actions in all animal models and CJ-033466 may stimulate the motor activity of the stomach in human. CJ-033466 could fill up the unmet needs after the withdrawal of cisapride from the clinical use.

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Acknowledgements I would like to express my gratitude to Professor Shingo Yano of Department of Molecular Pharmacology and Pharmacotherapeutics, Graduate School of Pharmaceutical Sciences, Chiba University for his supervisions, suggestions, kindness and continuous encouragement. I am sincerely grateful to Dr. Kohtaro Taniyama while he was a professor of Department of Pharmacology, Graduate School of Biomedical Sciences, Nagasaki University for the fruitful collaboration and his helpful advice. I would like to thank to Pfizer colleagues, Dr. Mitsuhisa Kawai, Mr. Naoji Kimura, Ms. Megumi Mitsuishi, Mr. Hiroki Mori, Mr. Yasuo Ochi, Dr. Minoru Sakakibara, Ms. Keiko Suzuki, Mr. Nobuyuki Takahashi in Nagoya and Dr. Jeremy Gale in Sandwich, UK for their kindness, support and assistance rendered to me during the execution of this research work.

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List of Publications (Main Thesis Publications) 1. Komada T., Yano S.: Pharmacological characterization of

5-hydroxytryptamine-receptor subtypes in circular muscle from the rat stomach. Biol. Pharm. Bull. (in press)

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Referees This thesis for the doctorate was judged by the following referees nominated in the Graduate School of Medical and Pharmaceutical Sciences, Chiba University. Referees: Toshihiko Murayama, Ph.D. (Pharm. Sci.) Professor of Chiba University (Graduate School of Pharmaceutical Sciences) Chief referee Toshiharu Horie, Ph.D. (Pharm. Sci.) Professor of Chiba University (Graduate School of Pharmaceutical Sciences) Haruaki Nakaya, Ph.D. (Med. Sci.) Professor of Chiba University (Graduate School of Medicine)

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elucidation for gastrokinetic effects of a novel 5-ht...

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